Humanized antibodies specific for ICAM related protein

ABSTRACT

DNA sequences encoding a novel human intercellular adhesion molecule polypeptide (designated &#34;ICAM-R&#34;) and variants thereof are disclosed along with methods and materials for production of the same by recombinant procedures. Binding molecules specific for ICAM-R and variants thereof are also disclosed as useful in both the isolation of ICAM-R from natural cellular sources and the modulation of ligand/receptor binding biological activities of ICAM-R. More specifically, humanized antibodies specific for ICAM-R proteins are disclosed.

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/102,852, filed Aug. 5, 1993 now abandoned which is in turn acontinuation-in-part of U.S. patent application Ser. No. 08/009,266,filed Jan. 22, 1993 now abandoned and International Application No.PCT/US93/00787, filed Jan. 26, 1993; which are in turncontinuations-in-part of U.S. patent application Ser. No. 07/894,061,filed Jun. 5, 1992 now abandoned; which is in turn acontinuation-in-part of U.S. patent application Ser. No. 07/889,724,filed May 26, 1992 now abandoned; which is in turn acontinuation-in-part of U.S. patent application Ser. No. 07/827,689,filed Jan. 27, 1992 now abandoned.

FIELD OF THE INVENTION

The present invention relates generally to cellular adhesion moleculesand more particularly to the cloning and expression of DNA encoding aheretofore unknown human polypeptide designated "ICAM-R" which possessesstructural relatedness to the intercellular adhesion molecules ICAM-1and -2.

BACKGROUND OF THE INVENTION

Research spanning the last decade has significantly elucidated themolecular events attending cell-cell interactions in the body,especially those events involved in the movement and activation of cellsin the immune system. See generally, Springer, Nature, 346: 425-434(1990). Cell surface proteins, and especially the so-called CellularAdhesion Molecules ("CAMs") have correspondingly been the subject ofpharmaceutical research and development having as its goal interventionin the processes of leukocyte extravasation to sites of inflammation andleukocyte movement to distinct target tissues. The isolation andcharacterization of cellular adhesion molecules, the cloning andexpression of DNA sequences encoding such molecules, and the developmentof therapeutic and diagnostic agents relevant to inflammatory processes,viral infection and cancer metastasis have also been the subject ofnumerous U.S. and foreign applications for Letters Patent. See Edwards,Current Opinion in Therapeutic Patents, 1(11): 1617-1630 (1991) andparticularly the published "patent literature references" cited therein.

Of fundamental interest to the background of the present invention arethe prior identification and characterization of certain mediators ofcell adhesion events, the "leukointegrins," LFA-1, MAC-1 and gp 150.95(referred to in WHO nomenclature as CD18/CD11a, CD18/CD11b, andCD18/CD11c, respectively) which form a subfamily of heterodimeric"integrin" cell surface proteins present on B lymphocytes, T lymphocytesmonocytes and granulocytes. See, e.g., Table 1 of Springer, supra, atpage 429. Also of interest are other single chain adhesion molecules(CAMs) that have been implicated in leukocyte activation, adhesion,motility and the like, which are events attendant the inflammatoryprocess. For example, it is presently believed that prior to theleukocyte extravasation which characterizes inflammatory processes,activation of integrins constitutively expressed on leukocytes occursand is followed by a tight ligand/receptor interaction between theintegrins (e.g., LFA-1) and one or both of two distinct intercellularadhesion molecules (ICAMs) designated ICAM-1 and ICAM-2 which areexpressed on blood vessel endothelial cell surfaces and on otherleukocytes.

Like the other CAMs characterized to date, e.g., vascular adhesionmolecule (VCAM-1) as described in PCT WO 90/13300 published Nov. 15,1990; and platelet endothelial cell adhesion molecule (PECAM-1) asdescribed in Newman et al., Science, 247: 1219-1222 (1990) and PCT WO91/10683 published Jul. 25, 1991!, ICAM-1 and ICAM-2 are structurallyhomologous to other members of the immunoglobulin gene superfamily inthat the extracellular portion of each is comprised of a series ofdomains sharing a similar carboxy terminal motif. A "typical"immunoglobulin-like domain contains a loop structure usually anchored bya disulfide bond between two cysteines at the extremity of each loop.ICAM-1 includes five immunoglobulin-like domains; ICAM-2, which differsfrom ICAM-1 in terms of cell distribution, includes two such domains;PECAM-1 includes six; VCAM includes six or seven, depending on splicevariations, and so on. Moreover, CAMs typically include a hydrophobic"transmembrane" region believed to participate in orientation of themolecule at the cell surface and a carboxy terminal "cytoplasmic"region. Graphic models of the operative disposition of CAMs generallyshow the molecule anchored in the cell membrane at the transmembraneregion with the cytoplasmic "tail" extending into the cell cytoplasm andone or more immunoglobulin-like loops extending outward from the cellsurface.

A variety of therapeutic uses have been projected for intercellularadhesion molecules, including uses premised on the ability of ICAM-1 tobind human rhinovirus. European Patent Application 468 257 A publishedJan. 29, 1992, for example, addresses the development of multimericconfigurations and forms of ICAM-1 (including full length and truncatedmolecular forms) proposed to have enhanced ligand/receptor bindingactivity, especially in binding to viruses, lymphocyte associatedantigens and pathogens such as Plasmodium falciparun.

In a like manner, a variety of uses have been projected for proteinsimmunologically related to intercellular adhesion molecules. W091/16928,published Nov. 14, 1991, for example, addresses humanized chimericanti-ICAM-1 antibodies and their use in treatment of specific andnon-specific inflammation, viral infection and asthma. Anti-ICAM-1antibodies and fragments thereof are described as useful in treatment ofendotoxic shock in W092/04034, published Mar. 19, 1992. Inhibition ofICAM-1 dependent inflammatory responses with anti-ICAM-1 anti-idiotypicantibodies and antibody fragments is addressed in W092/06119, publishedApril 16, 1992.

Despite the fundamental insights into cell adhesion phenomena which havebeen gained by the identification and characterization of intercellularadhesion proteins such as ICAM-1 and lymphocyte interactive integrinssuch as LFA-1, the picture is far from complete. It is generallybelieved that numerous other proteins are involved in inflammatoryprocesses and in targeted lymphocyte movement throughout the body. Quiterecently, for example, Springer and his co-workers postulated theexistence of a third counter-receptor for LFA-1 de Fougerolles et al.,J. Exp. Med., 174: 253-267 (1991)! and subsequently reported success inimmunoprecipitating a "third" ICAM ligand, designated "ICAM-3" deFougerolles, et al., J. Exp. Med., 175: 185-190 (1992)!. This moleculewas reported to bind soluble LFA-1 and to be highly expressed by restinglymphocytes, monocytes and neutrophils. Unlike ICAM-1 and ICAM-2,however, the new ligand was not found to be expressed by endothelialcells. The immunoprecipitated product was noted to display a molecularweight of about 124,000 and to be heavily glycosylated, as revealed by adrop in apparent molecular weight to about 87,000 upon N-glyanasetreatment. More recently, another research group described a cDNAsequence for a counter-receptor for LFA-1 which was also designated"ICAM-3" see Fawcett et al., Nature, 360: 481-484 (1992)!. Even morerecently, two articles were published by Springer and his co-workers deFougerolles et al., J. Exp. Med., 177: 1187-1192 (1993) and Juan et al.,Eur. J. Immunol., 23: 1508-1512 (1993)! which respectively report theamino acid sequence for ICAM-3 as being identical to that of ICAM-R andnote the identity of ICAM-3 to the differentiation antigen CDw50 basedon patterns of immunological reactivity of antibodies specific for eachprotein.

There thus continues to be a need in the art for the discovery ofadditional proteins participating in human cell-cell interactions andespecially a need for information serving to specifically identify andcharacterize such proteins in terms of their amino acid sequence.Moreover, to the extent that such molecules might form the basis for thedevelopment of therapeutic and diagnostic agents, it is essential thatthe DNA encoding them be elucidated. Such seminal information wouldinter alia, provide for the large scale production of the proteins,allow for the identification of cells naturally producing them, andpermit the preparation of antibody substances or other novel bindingproteins specifically reactive therewith and/or inhibitory ofligand/receptor binding reactions in which they are involved.

BRIEF SUMMARY

In one of its aspects, the present invention provides purified andisolated polynucleotides (e.g., DNA sequences and RNA transcriptsthereof, both sense and antisense strands) encoding a novel humanpolypeptide, "ICAM-R," as well as polypeptide variants (includingfragments and analogs) thereof which display one or more ligand/receptorbinding biological activities and/or immunological properties specificto ICAM-R. ICAM-R-specific ligand/receptor binding biological activitiesencompass interactions of both the ICAM-R extracellular and cytoplasmicdomains with other molecules (e.g., in processes of cell-cell adhesionand/or signal transduction). Preferred DNA sequences of the inventioninclude genomic and cDNA sequences as well as wholly or partiallychemically synthesized DNA sequences. Biological replicas (i.e., copiesof isolated DNA sequences made in vivo or in vitro) of DNA sequences ofthe invention are contemplated. Also provided are autonomouslyreplicating recombinant constructions such as plasmid and viral DNAvectors incorporating ICAM-R sequences and especially vectors whereinDNA encoding ICAM-R or an ICAM-R variant is operatively linked to anendogenous or exogenous expression control DNA sequence.

According to another aspect of the invention, host cells, especiallyunicellular host cells such as procaryotic and eucaryotic cells, arestably transformed with DNA sequences of the invention in a mannerallowing the desired polypeptides to be expressed therein. Host cellsexpressing such ICAM-R and ICAM-R variant products can serve a varietyof useful purposes. To the extent that the expressed products are"displayed" on host cell surfaces, the cells may constitute a valuableimmunogen for the development of antibody substances specificallyimmunoreactive with ICAM-R and ICAM-R variants. Host cells of theinvention are conspicuously useful in methods for the large scaleproduction of ICAM-R and ICAM-R variants wherein the cells are grown ina suitable culture medium and the desired polypeptide products areisolated from the cells or from the medium in which the cells are grown.

Novel ICAM-R and ICAM-R variant products of the invention may beobtained as isolates from natural cell sources, but are preferablyproduced by recombinant procedures involving host cells of theinvention. The products may be obtained in fully or partiallyglycosylated, partially or wholly de-glycosylated, or non-glycosylatedforms, depending on the host cell selected for recombinant productionand/or post-isolation processing.

Products of the invention include monomeric and multimeric polypeptideshaving the sequence of amino acid residues numbered -29 through 518 asset out in SEQ ID NO: 1 herein. As explained in detail infra, thissequence includes a putative signal or leader sequence which precedesthe "mature" protein sequence and spans residues -29 through -1,followed by the putative mature protein including, in order, fiveputative immunoglobulin-like domains (respectively spanning aboutresidues 1 to 90, 91 to 187, 188 to 285, 286 to 387, and 388 to 456), ahydrophobic "transmembrane" region extending from about residue 457 toabout residue 481 and a "cytoplasmic" region constituting the balance ofthe polypeptide at its carboxy terminus. Based on amino acidcomposition, the calculated molecular weight of the mature proteinlacking glycosylation or other post-translational modification isapproximately 52,417. ICAM-R variants of the invention may comprisewater soluble or insoluble monomeric, multimeric or cyclic ICAM-Rfragments which include all or part of one or more of the domain regionsspecified above and having a biological or immunological property ofICAM-R including, e.g., the ability to bind to a binding partner ofICAM-R and/or inhibit binding of ICAM-R to a natural binding partner.ICAM-R variants of the invention may also comprise polypeptide analogswherein one or more of the specified amino acids is deleted or replaced:(1) without loss, and preferably with enhancement, of one or morebiological activities or immunological characteristics specific forICAM-R; or (2) with specific disablement of a particular ligand/receptorbinding function. Analog polypeptides including additional amino acid(e.g., lysine or cysteine) residues that facilitate multimer formationare contemplated.

Also comprehended by the present invention are antibody substances(e.g., monoclonal and polyclonal antibodies, antibody fragments, singlechain antibodies, chimeric antibodies, CDR-grafted antibodies and thelike) and other binding proteins (e.g., polypeptides and peptides) whichare specific (i.e., non-reactive with the ICAM-1 and ICAM-2intercellular adhesion molecules to which ICAM-R is structurallyrelated) for ICAM-R or ICAM-R variants. Antibody substances can bedeveloped using isolated natural or recombinant ICAM-R or ICAM-Rvariants or cells expressing such products on their surfaces.Specifically illustrating antibodies of the present invention are themonoclonal antibodies produced by the hybridoma cell lines designated26E3D-1, 26I8F-2, 26I10E-2, 26H11C-2 which were deposited with theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852, on Jun. 2, 1992 as Accession Nos. HB 11054, HB11056, HB 11053, and HB 11055, respectively, in support of U.S. Ser. No.07/894,061; the hybridoma cell line designated 43H7C which was depositedwith the ATCC on Dec. 16, 1992 as Accession No. HB 11221 and thehybridoma cell lines designated 42C5H and 42D9B which were depositedwith the ATCC on Jan. 15, 1993 as Accession Nos. HB 11235 and HB 11236,respectively, in support of U.S. Ser. No. 08/009,266; the hybridoma celllines 46D7E and 46I12H which were deposited with the ATCC on Jan. 7,1993 as Accession Nos. HB 11232 and HB 11231, respectively, also insupport of U.S. Ser. No. 08/009,266; and the hybridoma cell lines63E11D, 63G4D, 63H4C, 63H6H, 63I 1C and 63I6G which were deposited withthe ATCC on Jul. 15, 1993 as Accession Nos. HB 11405, HB 11409, HB11408, HB 11407, HB 11406 and HB 11404, respectively, in support of U.S.Ser. No. 08/102,852; and the hybridoma cell line 81K2F, which wasdeposited with the ATCC on Jul. 27, 1994, as Accession No. HB 11692 insupport of this application. Various distinguishing properties ofbinding proteins of the invention are illustrated by these antibodiesand are summarized in Table 11 of Example 21 herein. Such propertiesinclude the ability to modulate CD18-dependent binding (e.g., to LFA-1and α_(d) /CD-18) and CD18-independent binding (e.g., to VLA-4) ofICAM-R to cells and cell surface molecules as well as the ability tomodulate lymphocyte activation by SEA and/or alloantigen. Bindingproteins of the invention are additionally susceptible tocharacterization in terms of binding site structure (e.g., epitopesand/or sensitivity of binding properties to modifications in ICAM-Ramino acid sequence).

Binding proteins are useful, in turn, in compositions for immunizationas well as for purifying polypeptides of the invention and identifyingcells displaying the polypeptides on their surfaces. They are alsomanifestly useful in modulating (i.e., blocking, inhibiting orstimulating) ligand/receptor binding biological activities involvingICAM-R, especially those ICAM-R effector functions involved in specificand non-specific immune system responses. Anti-idiotypic antibodiesspecific for anti-ICAM-R antibody substances and uses of suchanti-idiotypic antibody substances in modulating immune responses arealso contemplated. Assays for the detection and quantification of ICAM-Ron cell surfaces and in fluids such as serum may involve, for example, asingle antibody substance or multiple antibody substances in a"sandwich" assay format.

The scientific value of the information contributed through thedisclosures of DNA and amino acid sequences of the present invention ismanifest. As one series of examples, knowledge of the sequence of a cDNAfor ICAM-R makes possible the isolation by DNA/DNA hybridization ofgenomic DNA sequences encoding ICAM-R and specifying ICAM-R expressioncontrol regulatory sequences such as promoters, operators and the like.DNA/DNA hybridization procedures carried out with DNA sequences of theinvention and under stringent conditions are likewise expected to allowthe isolation of DNAs encoding allelic variants of ICAM-R, otherstructurally related proteins sharing one or more of the biologicaland/or immunological properties specific to ICAM-R, and non-humanspecies (e.g., rodent) proteins homologous to ICAM-R. DNAs of theinvention are useful in DNA/RNA hybridization assays to detect thecapacity of cells to synthesize ICAM-R. Also made available by theinvention are anti-sense polynucleotides relevant to regulatingexpression of ICAM-R by those cells which ordinarily express the same.As another series of examples, knowledge of the DNA and amino acidsequences of ICAM-R makes possible the generation by recombinant meansof ICAM-R variants such as hybrid fusion proteins (sometimes referred toas "immunoadhesions") characterized by the presence of ICAM-R proteinsequences and immunoglobulin heavy chain constant regions and/or hingeregions. See, Capon et al., Nature, 337: 525-531 (1989); Ashkenazi etal., P.N.A.S. (USA), 88: 10535-10539 (1991); and PCT WO 89/02922,published Apr. 6, 1989. ICAM-R variant fusion proteins may also include,for example, selected extracellular domains of ICAM-R and portions ofother cell adhesion molecules.

The DNA and amino acid sequence information provided by the presentinvention also makes possible the systematic analysis of the structureand function of ICAM-R and definition of those molecules with which itwill interact on extracellular and intracellular levels. The idiotypesof anti-ICAM-R monoclonal antibodies of the invention are representativeof such molecules and may mimic natural binding proteins (e.g., peptidesand polypeptides) through which ICAM-R intercellular and intracellularactivities are modulated or by which ICAM-R modulates intercellular andintracellular events. Alternately, they may represent new classes ofmodulators of ICAM-R activities. Anti-idiotypic antibodies, in turn, mayrepresent new classes of biologically active ICAM-R equivalents.

In vitro assays for identifying antibodies or other compounds thatmodulate the activity of ICAM-R may involve, for example, immobilizingICAM-R or a natural ligand to which ICAM-R binds, detectably labellingthe nonimmobilized binding partner, incubating the binding partnerstogether and determining the effect of a test compound on the amount oflabel bound wherein a reduction in the label bound in the presence ofthe test compound compared to the amount of label bound in the absenceof the test compound indicates that the test agent is an inhibitor ofICAM-R binding.

Another type of assay for identifying compounds that modulate theinteraction between ICAM-R and a ligand involves immobilizing ICAM-R ora fragment thereof on a solid support coated (or impregnated with) afluorescent agent, labelling the ligand with a compound capable ofexciting the fluorescent agent, contacting the immobilized ICAM-R withthe labelled ligand in the presence and absence of a putative modulatorcompound, detecting light emission by the fluorescent agent, andidentifying modulating compounds as those compounds that affect theemission of light by the fluorescent agent in comparison to the emissionof light by the fluorescent agent in the absence of a modulatingcompound. Alternatively, the ICAM-R ligand may be immobilized and ICAM-Rmay be labelled in the assay.

Yet another method contemplated by the invention for identifyingcompounds that modulate the interaction between ICAM-R and a ligandinvolves transforming or transfecting appropriate host cells with a DNAconstruct comprising a reporter gene under the control of a promoterregulated by a transcription factor having a DNA-binding domain and anactivating domain, expressing in the host cells a first hybrid DNAsequence encoding a first fusion of part or all of ICAM-R and either theDNA binding domain or the activating domain of the transcription factor,expressing in the host cells a second hybrid DNA sequence encoding partor all of the ligand and the DNA binding domain or activating domain ofthe transcription factor which is not incorporated in the first fusion,evaluating the effect of a putative modulating compound on theinteraction between ICAM-R and the ligand by detecting binding of theligand to ICAM-R in a particular host cell by measuring the productionof reporter gene product in the host cell in the presence or absence ofthe putative modulator, and identifying modulating compounds as thosecompounds altering production of the reported gene product in comparisonto production of the reporter gene product in the absence of themodulating compound. Presently preferred for use in the assay are theADHI promoter, the lexA DNA-binding domain, the GAL4 transactivationdomain, the lacZ reporter gene, and yeast host cells.

A modified version of the foregoing assay may be used in isolating apolynucleotide encoding a protein that binds to ICAM-R by transformingor transfecting appropriate host cells with a DNA construct comprising areporter gene under the control of a promoter regulated by atranscription factor having a DNA-binding domain and an activatingdomain, expressing in the host cells a first hybrid DNA sequenceencoding a first fusion of part or all of ICAM-R and either the DNAbinding domain or the activating domain of the transcription factor,expressing in the host cells a library of second hybrid DNA sequencesencoding second fusions of part or all of putative ICAM-R bindingproteins and the DNA binding domain or activating domain of thetranscription factor which is not incorporated in the first fusion,detecting binding of an ICAM-R binding protein to ICAM-R in a particularhost cell by detecting the production of reporter gene product in thehost cell, and isolating second hybrid DNA sequences encoding ICAM-Rbinding protein from the particular host cell.

The DNA sequence information provided by the present invention alsomakes possible the development, by homologous recombination or"knockout" strategies see, e.g., Kapecchi, Science, 244:1288-1292(1989)!, of rodents that fail to express a functional ICAM-R protein orthat express a variant ICAM-R protein. Such rodents are useful as modelsfor studying the activities of ICAM-R and ICAM-R modulators in vivo.

Modulators which affect the interaction between ICAM-R and LFA-1, α_(d)/CD18, VLA-4, tubulin, and the 14.3.3 family of proteins arespecifically contemplated as useful therapeutic compounds.

Inflammatory conditions which may be treated or monitored with ICAM-Rrelated products of the invention include conditions resulting from aresponse of the non-specific immune system in a mammal (e.g., adultrespiratory distress syndrome, multiple organ injury syndrome secondaryto septicemia, multiple organ injury syndrome secondary to trauma,reperfusion injury of tissue, acute glomerulonephritis, reactivearthritis, dermatosis with acute inflammatory components, stroke,thermal injury, hemodialysis, leukapheresis, ulcerative colitis, Crohn'sdisease, necrotizing enterocolitis, granulocyte transfusion associatedsyndrome, atherosclerosis and cytokine-induced toxicity) and conditionsresulting from a response of the specific immune system in a mammal(e.g., psoriasis, organ/tissue transplant rejection and autoimmunediseases including Raynaud's syndrome, autoimmune thyroiditis, EAE,multiple sclerosis, rheumatoid arthritis, diabetes, and lupuserythematosus). ICAM-R products of the invention may also be useful inmonitoring and treating asthma, tumor growth and/or metastasis, andviral infection (e.g., HIV infection).

In particular, disease processes in which T cell activation plays acentral and essential triggering role may be impacted beneficially byICAM-R related products of the invention described herein. Thetherapeutic use of ICAM-R analogs incorporating specific amino acidsubstitutions (e.g., analogs E37T or D231H) chosen to enhance ordiminish their specific immunomodulatory properties are useful in thisregard. Specific examples of T cell dependent diseases for which ICAM-Rrelated products may have utility include but are not limited to asthma,psoriasis, diabetes, graft vs. host disease, tissue transplantrejection, and multiple sclerosis. The use of products of the inventionto modulate diseases wherein macrophages play a central generative roleis also indicated. Moreover, monoclonal antibodies specific to ICAM-Rmay be used therapeutically either on their own or when conjugated toother moieties (e.g., toxins, radionuclides) to therapeutically targetand/or detect the presence of neovascularizing sites.

BRIEF DESCRIPTION OF THE DRAWING

Numerous other aspects and advantages of the present invention will beapparent upon consideration of the following detailed descriptionthereof, reference being made to the drawing wherein:

FIG. 1(A through G) depicts an isolated cDNA clone insert (SEQ ID NO: 2)derived from HL60 cells encoding ICAM-R and the deduced amino acidsequence (SEQ ID NO: 1) of an open reading frame therein;

FIG. 2(A through B) comprises bar graphs illustrating the results ofNorthern blot hybridization of transfected L cells using ICAM-R andICAM-1 DNA probes;

FIG. 3(A through F) presents photomicrographs depicting the results ofin situ hybridizations of transfected L cells using ICAM-R or ICAM-1 RNAprobes;

FIG. 4A comprises bar graphs illustrating the results of assays for theadhesion of PMA-stimulated or unstimulated lymphoblastoid cells frompatients with leukocyte adhesion deficiency to soluble ICAM-R in thepresence and absence of anti-CD18 antibody, while FIG. 4B comprises bargraphs illustrating the results of assays for the adhesion of variousother PMA-stimulated or unstimulated cell lines to soluble ICAM-R in thepresence and absence of anti-CD18 or anti-CD 11a antibody;

FIG. 5 illustrates in histogram format the results of FACS analyses ofindirect immunofluorescence staining of transfected L cells usingmonoclonal antibodies specific for ICAM-R, ICAM-1 or ICAM-2;

FIG. 6 is a diagram of three chimeric ICAM-R proteins utilized to mapepitopes of anti-ICAM-R monoclonal antibodies of the invention;

FIG. 7(A through B) presents bar graphs depicting the results ofactin-normalized Northern blot hybridization of human leukocyte celllines and umbilical cord endothelial cells using ICAM-R or ICAM-1 DNAprobes;

FIG. 8(A through B) comprises photographs of Western blots ofimmunoprecipitations of lysates from human cells lines using ICAM-Rspecific monoclonal antibodies;

FIG. 9(A through G) presents photomicrographs of immunohistologicstaining of various human tissues with an anti-ICAM-R monoclonalantibody;

FIG. 10 is a bar graph depicting the effects of anti-ICAM-R monoclonalantibodies on the stimulation of lymphocyte proliferation by anti-CD3antibodies;

FIG. 11(A through B) comprises bar graphs illustrating the effects ofanti-ICAM-R monoclonal antibodies on superantigen-induced proliferationof human peripheral blood lymphocytes, while FIG. 11C is a graphcomprising logistic dose response curves of the effects of anti-ICAM-Rmonoclonal antibodies on superantigen-induced proliferation of humanperipheral blood lymphocytes;

FIG. 12 is a bar graph depicting the effects of anti-ICAM-R monoclonalantibodies on alloantigen-induced T-cell proliferation; and

FIG. 13 is a bar graph illustrating the effect of anti-ICAM-R monoclonalantibodies on superantigen-induced proliferation of "memory" T cells;

FIG. 14 comprises a bar graph depicting the effect of anti-ICAM-Rmonoclonal antibodies on superantigen-induced proliferation of "resting"T cells;

FIG. 15 comprises a bar graph illustrating that crosslinking distinctICAM-R epitopes differentially affects ICAM-R association with thecytoskeleton.

FIG. 16 is a schematic depiction of the three-dimensional structure ofthe extracellular domain 1 of ICAM-R.

DETAILED DESCRIPTION

The present invention is illustrated by the following examples relatingto the isolation of a full length cDNA clone encoding ICAM-R from a cDNAlibrary derived from human HL60 promyelocytic cells (ATCC CCL 240) andto the expression of ICAM-R DNA in L cells. More particularly, Example 1addresses the design and construction of oligonucleotide probes for PCRamplification of ICAM related DNAs. Example 2 addresses the use of theprobes to amplify a genomic DNA fragment homologous to, but distinctfrom, DNAs encoding ICAM-1 and ICAM-2. Example 3 treats the screening ofcDNA libraries with the genomic fragment to isolate additional ICAM-Rcoding sequences. Example 4 refers to the further screening of cDNAlibraries to isolate a full length human cDNA encoding ICAM-R. Example 5provides a characterization of DNA and amino acid sequence informationfor ICAM-R, relates the structures thereof to ICAM-1 and ICAM-2,describes the chromosomal localization of the ICAM-R gene and describesthe isolation of human ICAM-R genomic sequences. Example 6 relates tothe development of mammalian host cells expressing ICAM-R. Example 7describes preliminary experiments indicative of ICAM-R participation inintercellular adhesion events involving CD18-dependent andCD18-idependent pathways. Example 8 presents experiments illustratinginhibition of cell adhesion to ICAM-R by ICAM-R derived peptides.Example 9 relates to the construction and expression of a solublevariant of human ICAM-R and various assays useful for identifying ICAM-Rligands and modulators of ICAM-R activities. Example 10 describes theconstruction and expression of ICAM-R variants having point mutations intheir extracellular domains. Example 11 describes the preparation andpreliminary characterization of anti-ICAM-R antibodies and thepreparation of Fab' fragments thereof. Example 12 relates to assaysdetermining the capability of ICAM-R specific monoclonal antibodies toinhibit binding of CD18⁺ cells to recombinant soluble human ICAM-R.Example 13 details the humanization of ICAM-R specific monoclonalantibodies of the invention. Example 14 relates to mapping of the ICAM-Repitopes recognized by the anti-ICAM-R monoclonal antibodies of theinvention. Examples 15, 16, 17 and 18 relate to assessment of thedistribution and biochemical characterization of ICAM-R polypeptide andRNA encoding the same in normal cells and tissues as well as in variouscell lines. Example 19 describes assays for the involvement of ICAM-R inhomotypic cell-cell adhesion. Example 20 addresses experimentsindicating that ICAM-R is involved in immune cellactivation/proliferation. Example 21 comprises a summary ofcharacteristics of ICAM-R specific monoclonal antibodies of theinvention. Example 22 describes experiments showing differentialphosphorylation of and cytoskeletal associations with the cytoplasmicdomain of ICAM-R. Examples 23 and 24 set out experiments characterizingthe interaction between ICAM-R and various cytoplasmic ligands utilizingdihybrid screening techniques. Example 25 describes the interactionbetween ICAM-R and LFA-1 while Examples 26 and 27 describe theinteraction between ICAM-R and α_(d) /CD18 and ICAM-R and VLA-4,respectively. Example 28 provides evidence that elevated levels ofsoluble ICAM-R are observed in human serum various immune-mediateddiseases. Example 29 describes various therapeutic applications ofsubject matter of the invention. The use of ICAM-R-specifc monoclonalantibodies of the invention to prevent development of graft versus hostdisease is described in Example 30. Example 31 details the isolation ofcanine and rabbit ICAM-R sequences and generation of reagents using thesequences that are useful in animal models of disease states.

EXAMPLE 1

Nucleic acid and amino acid alignments of individual sets of CAMs (e.g.,ICAM-1 and ICAM-2) did not manifest sufficient conservation betweenmolecules to yield information useful in the design of consensus-typeprobes for isolating related novel genes. The strategic focus ofattempts to isolate unknown DNAs encoding cellular adhesion moleculestherefore involved the development of degenerate consensusoligonucleotides representing putative spaced apart DNA sequences ofvarious known molecules and the use of these oligonucleotides as primersfor polymerase chain reaction (PCR) amplification of DNA replicas ofintermediate gene sequences which resemble, but are not identical to,the known DNAs. The starting point for oligonucleotide primer design wasthe notation that the amino acids in regions surrounding cysteines whichform immunoglobulin-like loops of certain CAMs are somewhat conserved.At the amino terminal side of the motif, the sequence:

SEQ ID NO: 3

G-X-X-(V or L or I)-X-(V or L or I)-X-C

is found, while at the carboxy terminal side of the motif, the sequence:

SEQ ID NO: 4

N-X-G-X-Y-X-C-X-(V or A)

is typical. See Hunkapiller et al., Nature, 323: 15-16 (1986); Williamset al, Ann. Rev. Immunol., 6: 381-405 (1988); and Newman et al, supra.!In and of themselves the two amino acid motifs are much too general anddo not allow the construction of degenerate sets of oligonucleotidesuseful as probes for unknown DNAs which might share the motif. In anattempt to solve this problem, each individual CAM sequence was splitinto a domain of sub files defined by the cysteine motif terminidescribed above. Subfiles were generated for each of the seven domainsof human vascular adhesion molecule (VCAM-1), the six domains of humanplatelet endothelial cell adhesion molecule (PECAM-1), the five domainsof ICAM-1, the two domains of ICAM-2, three of the four domains of bothhuman myeloglobin-related glycoprotein and human fibroblast growthfactor receptor, and the five domains of mouse neural cell adhesionmolecule (NCAM). All the subfiles were pooled and segregatedindependently from the CAM of origin using a multialignment homologycomputer algorithm designated "Multalin" Corpet, Nucleic Acids Research,16(22): 10881-10890 (1988)! providing a tree of alignment allowing theascertainment of consensus sequences around cysteine motifs. A consensussequence representing the amino terminal cysteine motif was determinedto be:

SEQ ID NO: 5

G-K-(N or S)-(L or F)-T-(L or I)-(R or E)-C

while the carboxy terminal consensus sequence was determined to be:

SEQ ID NO: 6

(D or E)-(H or D)-(H or G)-(G or H)-(A or R)-N-F-S-C.

Employing human preferences for codon usage to partially eliminatedegeneracy, three separate sets of degenerate oligonucleotides totaling1152 probes were generated for use as top strand PCR primers foramplification from a putative amino terminus of the motif. The specificdegenerate sequences of the three pools are set out below in IUPACnomenclature.

SEQ ID NO: 7

ATTCTGCAGGCAARAAYCTSACHMTBMGSTG

SEQ ID NO: 8

ATTCTGCAGGCAARAGYTTYACHMTBGARTG

SEQ ID NO: 9

ATTCTGCAGGCAARTCYTTYACHMTBGARTG

Each of the primers included a PstI restriction endonuclease recognitionsite (CTGCAG) to facilitate cloning of amplified products.

A total of 768 probes were designed as bottom strand primers as set outbelow in IUPAC nomenclature for amplification from a putative carboxyterminus of the motif. Each of these primers included an XbaIrecognition site (TCTAGA) to facilitate cloning of amplified products.

SEQ ID NO: 10

ATITCTAGARAARTTRGCSCCRTGRTSRTC

SEQ ID NO: 11

ATTTCTAGARAARTTSCKRTGSCCRTSKTC

Oligonucleotides were synthesized with an automated Applied Biosystems,Inc. (Foster City, Calif.) Model 394 DNA synthesizer using an 0.2micromolar scale synthesis program and employing beta-cyanoethylchemistry. Protective groups were then removed by heating at 55° C. forin excess of six hours. Oligonucleotides were then lyophilized todryness, rehydrated in TE (10 mM Tris, pH 7.0, 1mm EDTA) and desalted inTE by size exclusion chromatography with G25-150 Sephadex.

EXAMPLE 2

The two sets of probes whose design and synthesis are described inExample 1 were employed in PCR amplification procedures applied to ahuman genomic DNA template. Briefly put, PCR-generated fragments of asize similar to that of the immunoglobulin-like loop regions of ICAM-1and ICAM-2 were isolated, subcloned into Bluescript plasmid (Stratagene,La Jolla, Calif.) and screened both directly by sequencing andhybridization in arrays for homology to ICAM-2 DNA. Approximately 50% ofthe fragments were identical to ICAM-1 or ICAM-2 (except, of course, inthe regions of the degenerate primer). One subclone, designated 13-3C7,was found to have an open reading frame homologous to ICAM-1 and ICAM-2in the region of their respective second domains. It did not correspondto any known sequence present in the Genbank data base. The specificmanipulations leading up to the isolation of subclone 13-3C7 were asfollows.

The degenerate oligonucleotides were mixed to a final concentration of10 μg/ml in a PCR reaction to amplify human genomic DNA obtained eitherfrom peripheral blood leukocytes or Hela cells. The DNA amplificationwas performed in PCR buffer (2 mM MgCl₂, 25 mM KCl, 10 mM Tris pH 8.3)with 2 mM deoxynucleotides. After a 94° C. denaturation for 4 minutes,30 PCR cycles were performed with annealing at 60° C. for 2 minutes,elongation at 72° C. for 4 minutes and denaturation at 94° C. for 1minute. A DNA band migrating at about 0.2 kb was extracted from a 6%polyacrylamide gel by electroelution, digested by XbaI and Pst 1restriction enzymes, and ligated into the Bluescript vector(Stratagene). The plasmid was electroporated into XL 1-blue strains ofE.coli (Stratagene) and colonies were selected on X-gal IPTG,carbenicillin agarose plates. Single strand templates were obtained from6 white colonies after addition of M13K07 helper phage (Stratagene),carbenicillin, and kanamycin to a 2 ml culture of each colony. Forsequence analysis, the single strand templates were then sequenced usingthe Sanger method both by DNA automatic sequencing (Applied Biosystems)and with a sequenase kit (UCB, Belgium). Four sequences (clones 1.1,1.3, 1.4, 1.6) were obtained which were 184-185 base pairs (bp) long andwere 92-95% homologous to the second domain of ICAM-2. In addition, a182 bp long DNA sequence (clone 1.5) was obtained which contained aframeshift in the open reading frame of an ICAM-1-like domain along witha 66 bp DNA (clone 1.2) corresponding to a truncated immunoglobulin-likedomain.

The sequence of clones 1.6, 1.5, 1.2 was used to design threeoligonucleotide probes (RM16, RM15, RM12) that were used in subsequenttests to eliminate from further consideration additional coloniescontaining cDNAs that were highly homologous to the previous isolatedclones. The sequences of probes RM16, RM15 and RM12 are set out below.

Probe RM16 (SEQ ID NO: 12)

GAGACTCTGCACTATGAGACCTTCG

Probe RM15 (SEQ ID NO: 13)

CAGGTGATTCTCATGCAGAGTCCAGG

Probe RM12 (SEQ ID NO: 14)

CCGACATGCTGGTAAGTGTGTCCAA

In a second round of tests, new colonies were obtained from the originalPCR products that had been XbaI and Pst1 digested and from additionalPCR products that had been rendered blunt-ended by treatment with theKlenow fragment of polymerase I and subcloned by blunt-end ligation. Thecolonies containing the vector with an insert were selected oncarbenicillin L broth agarose plates containing X-gal and IPTG. Singlestrand templates were then synthesized in 96-well plates by growingindividual white colonies in 300 μl L broth, to which was added M13K07phage, carbenicillin and kanamycin. Ten μl of each template wastransferred with a pronging device to a nylon membrane, denatured andfixed with UV light. (Ten μl of each template were transferred to threedifferent nylon membranes for each 96-well plate.) OligonucleotidesRM16, RM15, RM12 were labelled by phosphorylation using λ-³² P!ATP. Thenylon membranes were pre-hybridized in 20% formamide, 5× SSC, 5×Denhardt's solution and 0.5% SDS for 3 hours at 42° C. then hybridizedovernight with the different radiolabelled oligonucleotide probes underthe same conditions. The membranes were then washed in 0.2× SSC, 0.5%SDS three times for 15 minutes each at room temperature then washed inthe same buffer at 37° C. for 15 minutes, rinsed in 2× SSC and exposed.Each template that did not hybridize with either of the threeoligonucleotide probes was further sequenced using the Sanger techniqueby DNA automatic sequencing and by sequenase kit. Using this technique,the 170 bp DNA sequence of a clone designated 13-3C7 was determined.

EXAMPLE 3

The cDNA insert of subclone 13-3C7 isolated in Example 2 was used as ahybridization probe to screen four different lambda phage cDNA librariesprepared from human spleen, human placenta (two libraries) and humanleukocyte cell line U937 (ATCC CRL 1593). Briefly summarized, onehundred and twenty positive clones were picked (from among theapproximately 1.6 million clones screened), subcloned, rescreened withthe 13-3C7 probe, and the rescreening positive were size selected forinserts of greater than approximately 500 bp by analytical PCR withprimers corresponding to the plasmid DNA flanking the insertion forDNAs. A 1.3 kb clone derived from U937 cDNA, designated clone 19C, wassequenced and revealed DNA regions encoding two immunoglobulin-likedomains separated by what appeared to be an intervening sequence(intron) resulting from improper or incomplete mRNA splicing prior tocDNA formation. The two regions displayed significant homology, butoverall distinctness, in comparison to domains 2 and 3 of ICAM-1 andless homology to domains 1 and 2 of ICAM-2.

The specific procedures leading up to isolation of clone 19C were asfollows. The four libraries were constructed in lambda gt10 phage(λgt10) using cDNA obtained from the U937 cell line, from the spleen ofa patient with chronic myelomonocytic leukemia and from human placenta.Exact match oligonucleotides designated 1 Hr-5' and 1Hr-3' were designedcorresponding to the 5' and 3' sides of the domain-like region ofsubclone 13-3C7 (including bases attributable to incorporation of theoriginal degenerate primer). The sequences of the 1 Hr-5' and 1 Hr-3'oligonucleotide primers are set out below.

Primer 1 Hr-5' (SEQ ID NO: 15)

GACCATGAGGTGCCAAG

Primer 1 Hr-3' (SEQ ID NO: 16)

ATGGTCGTCTCTGCTGG

Using these oligonucleotides in a PCR reaction with the 13-3C7 inserttemplate and ³² P-dCTP, a 148 bp long DNA probe was generated. The cDNAlibraries were plated and transferred to nylon membranes. The membraneswere pre-hybridized in 40% formamide, 5× SSC, 5× Denhardt's, 0.5% SDS at42° C. for at least 15 minutes, then hybridized overnight with the probein the same buffer at 42° C. The membranes were washed several times atroom temperature in 2× SSC and exposed. Most of the phage plaques thathybridized with the probe were derived from the U937 cDNA library. Thesephages were further purified and tested by PCR (using 1 Hr-5' and 1Hr-3' as primers) for the presence of the domain inside the cDNA clones.The phage were also tested by PCR to determine the length of the clonesand the location of the domain within the cDNA fragment (using acombination of 13-3C7 specific primers and primers homologous toflanking λgt10 vector sequences). Two clones were selected. Clone 1F was0.7 kb long and clone 19C was 1.3 kb long. These cDNAs were digestedwith EcoRI and subcloned in the Bluescript vector. In addition, thelargest cDNA (clone 19C) was sonicated to obtain small pieces which weresub-cloned into Bluescript for sequencing. By homology with the ICAM-1molecule, clone 19C cDNA contains 2 regions having homology to domains 2and 3 of ICAM-1, respectively, with an intervening sequence of unrelatedDNA. Hereinafter, these DNA regions are referred to as domains 2 and 3of ICAM-R.

EXAMPLE 4

The 1.3 kb (clone 19C) DNA isolated in Example 3 and having regionsencoding immunoglobulin-like loops resembling domains 2 and 3 of ICAM-1was then employed to generate a probe for the screening of additionalcDNA libraries in an attempt to isolate a full length cDNA clone.Briefly, the domain 2 and 3 regions within clone 19C were each amplifiedby PCR using unique probes designated to match respective amino (5') andcarboxy (3') terminal portions of the domains. These amplified DNAs, inturn, provided probes for screening of cDNA libraries derived from: (1)the HL60 myelomonocytic cell line; (2) lipopolysaccharide-activatedhuman monocytes; (3) HUT-78 T-cells (ATCC T1B161); and (4) activatedperipheral blood leukocytes. The latter two libraries yielded nopositive upon rescreening. Positives derived from HL60 and monocyte cDNAlibraries were then screened with a probe representing domain 2 ofICAM-1 DNA (GenBank, Accession No. 22634) in order to eliminate ICAM-1clones. A single phagmid clone derived from lambda 345 and designatedpVZ-147, repeatedly tested positive for hybridization with the probe(s)based on the DNA isolated in Example 4 and negative for hybridizationwith the ICAM-1 DNA probe. The approximately 1.7 kb insert from clonepVZ-147 was isolated and sequenced to provide the 1781 bp sequence setout in SEQ ID NO: 2. The deduced amino acid sequence of the polypeptideencoded by this DNA is set out in SEQ ID NO: 1. The polypeptide wasdesignated "ICAM-R" on the basis of its structural relatedness to ICAM-1and ICAM-2. The DNA and deduced amino acid sequences of ICAM-R werepublished after the priority dates of this application in Vazeux et al.,Nature, 360: 485-488 (1992). The open reading frame of the DNA sequenceof ICAM-3 published after the priority dates of this application inFawcett et al., supra, differs at two nucleotide positions from thecoding region of the DNA sequence of ICAM-R presented in FIG. 1(Athrough G) herein. (See nucleotide positions 194 and 1275.)

The specific manipulations involved in the isolation of lambda phageclone pVZ147 are as follows. All cDNA libraries were constructed inλgt10 except for the HL60 library which cloned into phage lambda 345.Oligonucleotides for use in library screening and rescreening had thefollowing sequences.

Probe IHr2-5' (SEQ ID NO: 17)

TTCACCCTGCGCTGCCAA

Probe IHr2-3' (SEQ ID NO: 18)

AAAGGGGCTCCGTGGTCG

Probe IHr 3-5' (SEQ ID NO: 19)

CCGGTTCTTGGAGGTGGAA

Probe IHr 3-3' (SEQ ID NO: 20)

CATGACTGTCGCATTCAGCA

Probe Icam 1-5 (SEQ ID NO: 21)

GCAAGAACCTTACCCTAC

Probe Icam 1-3 (SEQ ID NO: 22)

GAAATTGGCTCCATGGTGA

Probes IHr 2-5' and IHr 2-3' were employed in a PCR amplification using³² P-dCTP on the clone 19C template to generate a domain 2 specificprobe for CDNA screening. Likewise, probes IHr 3-5' and IHr 3--3' wereemployed to generate a domain 3 specific probe. Finally, probes Icam 1-5and Icam 1-3 were employed to amplify an ICAM-1 segment probecorresponding to bases 440 through 609 of the ICAM-1 cDNA sequence(GenBank, Accession No. 22634), i.e., the ICAM-1 second domain.

The cDNA libraries were plated, transferred on nylon membranes,hybridized with the domain 2 probe (derived from clone 19C) in 40%formamide, 5× SSC, 5× Denhardt, 0.5% SDS and washed as described above.All the plaques that hybridized with the domain 2 probe were derivedfrom the monocyte and HL60 libraries. These phage plaques were purifiedby dilution, plating, transfer and hybridization with the domain 2probe. To further characterize the CDNA clones, each plaque that hadhybridized with the domain 2 probe was grown on an array in triplicate,transferred to a nylon membrane and hybridized under higher stringencyconditions (50% formamide, 5× SSC, 5× Denhardt, 0.5% SDS) with threedifferent probes: the domain 2 probe; the domain 3 probe, and the ICAM-1second domain probe. Five clones were found in the HL60 library and 2clones in the monocyte library which hybridized with both domain 2 anddomain 3 probes and not with the ICAM-1 second domain probe. A sixthclone from the HL60 library hybridized only with domain 2 probe and didnot hybridize with either domain 3 or with ICAM-1 second domain. ThecDNAs of the 6 clones from the HL60 library were further analyzed. Thephages were tested by PCR for the presence of properly spliced cDNAusing oligonucleotide primers corresponding to the 5' extremity(IHr2-5') of domain 2 and to the 3' extremity (IHr3--3') of domain 3.The clones were also tested by PCR for length and location of thedomains inside the clones. The cDNA plasmids were extracted and cyclizedfrom phage lambda 345 by digestion with SfiI and self-ligation. Tofacilitate making single strand templates and sequencing in bothorientations, each cDNA was also subcloned in Bluescript SK+vector(Stratagene). Plasmid pVZ147 was determined to include the entire ICAM-Rcoding sequence in a single open reading frame.

EXAMPLE 5

A. Characterization of the ICAM-R Polypeptide

FIG. 1 (A through G) graphically illustrates the sequence of the humancDNA insert of the lambda phage clone pVZ 147 isolated in Example 4,above. The total of 1781 bp shown are as set out in SEQ ID NO: 2. Thededuced amino acid sequence of the ICAM-R polypeptide as set out in SEQID NO: 1 is graphically subdivided in FIG. 1(A through G) into thefollowing regions:

(1) A putative signal or leader sequence is illustrated preceding thesequence of the "mature" protein and spanning amino acids designated -29through -1. Determination of whether the translation product is actuallyinitiated at -29 or -26 will be provided by amino acid sequencing ofintercellular expression products. The designation of the first residueof the mature protein was based on generalized analogy to amino acids(and corresponding bases) for residues of secreted human proteins in theregion of the junction of the mature protein and leader sequences.Confirmation of the actual initial residue of the mature protein awaitssequencing of a secreted recombinant product or, e.g., an immunopurifiednatural product.

(2) Within the mature protein spanning residues +1 through 518, fiveputative immunoglobulin-like loop regions are shown (white on black)bounded by cysteines within the five putative immunoglobulin-likedomains (shown in boxes). Note that in the first domain (residues 1through 91), cysteine residues potentially significant to loop formationare present at positions 24, 28, 67 and 71. Each of the remainingputative loops has a single relevant cysteine at each of its ends.

(3) Also within the mature protein, a putative hydrophobic"transmembrane" region is illustrated with dashes connecting residues457 through 481 which follow the fifth immunoglobulin-like domain. Aputative carboxy terminal "cytoplasmic" region constitutes residues 482through 518.

(4) Potential N-linked glycosylation sites characterized by theconsensus sequence, Aspargine-X-(Serine or Threonine)! are indicatedwith an asterisk. Potential O-linked glycosylation sites occur at anyserine or threonine residue.

A comparison was made between the amino acid sequence (SEQ ID NO: 1) ofICAM-R and the published 537 residue amino acid sequence of ICAM-1(GenBank Accession No. 22634; cf, FIG. 8 of European Patent Application0 289 949 published Nov. 11, 1988). This comparison revealed 249 matcheswithin the aligned 537 residues, indicating an overall amino acididentity of 48% between the two polypeptides. The highest percentage ofmatches was noted to be present between domains 2 and 3 of ICAM-1 andputative domains 2 and 3 of ICAM-R. Likewise the alignment of SEQ ID NO:1 with the published 295 residues of the amino acid sequence of ICAM-2(GenBank accession No. 22635; cf, FIG. 2 of European Patent Application0 387 668 published Sep. 19, 1990) revealed 78 matches among the 282aligned residues, for a 27% overall identity of amino acids in onepossible alignment. The cytoplasmic domain of ICAM-R was found to be 20%identical to the cytoplasmic domain of ICAM-1 and 34% identical to thecytoplasmic domain of ICAM-2 in one possible alignment.

B. Characterization of ICAM-R DNA

A comparative alignment of the human ICAM-R DNA sequence (SEQ ID NO: 2)was made with the published DNA sequences of ICAM-1 and ICAM-2, supra. Atotal of 677 matches were noted among the 1623 aligned bases of ICAM-Rand ICAM-1 providing an overall identity of 41%. A 42% identity (484matches) between the aligned 1136 bases of ICAM-R and ICAM-2 DNAs wasnoted.

Reference points in the FIG. 1 (A through G) DNA having "historical"significance to the isolation of the ICAM-R gene include the following:

(a) bases 420 through 567 correspond to the subclone 13-3C7 isolated inExample 2;

(b) bases 373 through 663 correspond to the immunoglobulin-like domain 2localized in clone 19C of Example 3 (with bases 418 through 435 and 561through 578, respectively corresponding to probes IHr2-5' and IHr2-3'employed for PCR amplification of domain 2 to provide one of theoligonucleotide probes for use in Example 4); and

(c) bases 664 through 957 correspond to the immunoglobulin-like domain 3localized on clone 19C of Example 3 (with bases 699 through 717 and 800through 819, respectively corresponding to probes IHr3-5' and IHr3--3'employed for PCR amplification of domain 3 to provide anotheroligonucleotide probe for use in Example 4.

C. Chromosomal Localization of Sequences Encoding Human ICAM-R

An ICAM-R specific DNA probe was utilized in the methods described inCannizzaro et al., Cancer Res., 51: 3818-3820 (1991) to determine thatthe human ICAM-R encoding sequences are located on chromosome 19 withprimary localization to the short (p) arm region.

D. Cloning of Genomic ICAM-R Sequences

Human ICAM-1 and -R have been mapped to the same region of chromosome19. Therefore, the human P1 Genomic library of Genome Systems Inc. (St.Louis, Mo.) was screened with human ICAM-1 oligonucleotides:

H-1/D3(S) (SEQ ID NO: 23)

CCGGGTCCTAGAGGTGGACACGCA and

H-1/D3(AS) (SEQ ID NO: 24)

TGCAGTGTCTCCTGGCTCTGGTTC,

designed to amplify a 230 bp fragment of ICAM-1 domain 3. Two clones(1566 and 1567) containing 75-95 kb genomic DNA inserts were analyzed.Plasmid DNA from each clone was digested with BamHI and blotted ontonylon membranes. Southern blots were hybridized under either lowstringency (30% formamide) or high stringency (60% formamide) at 42° C.with an ICAM-R domain 1 through 4 radiolabelled probe (otherconstituents of the hybridization solution were as described in Example6A). The low stringency hybridization series was washed at roomtemperature in 2× SSPE, 0.1% SDS. The high stringency hybridizationseries was washed at 65° C. in 0.2× SSPE, 0.1% SDS. The washed membraneswere exposed to X ray film for 3.5 hours. ICAM-R genomic sequences weredetermined to be located on 4.0 kb and 1.5 kb BamHI fragments. TheICAM-R fragments were subcloned into pBS+ (Stratagene) and theiridentity confirmed by limited sequence analysis. The genomic sequenceinformation obtained for ICAM-R corresponds to the third domain of theprotein.

Fragments of genomic DNA upstream of the leader sequence of human ICAM-Rwere also cloned and sequenced. P1 plasmid 1566 (which is described inthe foregoing paragraph) containing ICAM-R genomic sequences wasdigested with a number of restriction enzymes, run on an agarose gel andtransferred to nylon membranes. The membranes were then hybridized witha radiolabeled oligonucleotide derived from the leader sequence of humanICAM-R H3L5' (S) corresponding to nucleotides 16 to 33 of SEQ ID NO: 2!.This involved prehybridization in a solution containing 5× SSPE, 10×Denhardts and 1% SDS at 42° C. for 3 hours followed by the addition ofthe radiolabeled oligonucleotide. The hybridization continued at 42° C.overnight. The blot was then washed in 2× SSPE with 0.1% SDS at roomtemperature for 10-20 minutes and exposed to X-ray film for a few hours.A 2.4 kb ApaI fragment was identified in this manner. To subclone the2.4 kb ApaI fragment, DNA from the P1 clone was gel purified, cut withApaI, and ligated into pbluescript (Stratagene) that had been cut withApaI. A positive clone (P1-Apa) was identified with another round ofhybridization using oligonucleotide H3L5' (S) and sequenced in itsentirety. The leader sequence was located 1260 bp from the 5' end of thePI-Apa clone.

To obtain further sequence information in the upstream region of humanICAM-R, another overlapping fragment was cloned. A restriction map ofthe P1-Apa clone revealed an internal PstI site near the 5' end. The P1clone 1566 was digested with PstI and the fragments subcloned intopbluescript. The colonies containing the overlapping PstI fragment wereidentified by hybridization with oligonucleotides corresponding to the5' end of the P1-Apa clone. The P1-Pst clone was sequenced and found tooverlap with the P1-Apa clone, providing an additional 270 bp ofsequence information further upstream from the leader of ICAM-R. Thecomposite sequence upstream of the leader is set out in SEQ ID NO: 117.The sequence is 1600 bp long, the last seventy-eight nucleotides ofwhich encode the leader of human ICAM-R.

The precise location of promoter elements within the upstream regulatoryregion remains to be determined. Analysis of the region does not revealthe presence of a TATA box usually associated with basal transcriptioninitiation. To determine which regions of the upstream region contributeto promoter activity, the upstream region (or subfragments thereof) issubcloned upstream of a reporter gene that lacks a promoter. Expressionin lymphoid and nonlymphoid cell lines is then tested to characterizethe basal and inducible components of the promoter.

EXAMPLE 6

Human ICAM-R cDNA was transfected into L-M(TK⁻) mouse cells (ATCC CCL1.3) and the cells were assayed for expression of ICAM-R by Northernblot and in situ hybridization.

A. Transfection of ICAM-R DNA

The full length human ICAM-R cDNA insert of pVZ-147 (Example 4) and asmall portion of the phagmid vector 3' to the cDNA insert was excisedusing restriction enzymes NotI and XbaI and ligated into commercialplasmid pCDNA1-neo (Invitrogen Inc., San Diego, CA) cut with NotI andXbaI. The resulting plasmid, designated pCDNA1-neo-ICAM-R, wastransfected into mouse L cells by the calcium phosphate precipitationmethod described in Chen et al., Molecular and Cellular Biology, 7:2745-2748 (1987). ICAM-1 DNA (construct pCDNA-neo-ICAM-1) was alsotransfected into mouse L cells as a control. A cDNA fragment containingthe complete ICAM-1 protein coding region was ligated into plasmidpCDNA1-neo and transfected into L cells by the calcium phosphateprecipitation method. Following selection for neomycin resistance,individual ICAM-R or ICAM- 1 transfectants were subcloned using cloningcylinders (Bellco Glass Inc., Vineland, N.J.). The clones expressing thehighest level of ICAM-R and ICAM-1 protein were then sorted on acell-sorter.

Constructs pCDNA-neo-ICAM-R and pCDNA-neo-ICAM-1 were also transfectedinto CV-1 cells by the calcium phosphate precipitation method. Theclones expressing high levels of ICAM-R and ICAM-1 were selected asdescribed above for L cell tranfectants. Based on FACs analysis withICAM-R and ICAM-1 specific antibodies the level of protein expressionwas higher with CV-1 transfectants then with the mouse LTKtransfectants.

B. Northern Blot Hybridizations

Following transfection of full length ICAM-R or ICAM-1 cDNAs into mouseL cells, specific expression of the corresponding mRNAs in transfectedand untransfected L cells was determined by Northern blot hybridizationwith ³² P-labelled ICAM-R or ICAM-1 DNA probes. Transfectants were grownin log phase, then centrifuged and washed two times with 150 mM NaCl.The pellet was resuspended in 3.5 ml GIT (guanidinium isothiocyanate)buffer, then sheared in a polytron mixer for 20 seconds. After adding1.7 ml CsCl buffer to an ultracentrifuge tube, the GIT/RNA mix waslayered on top. Samples were spun at 35 K (179,000×g), 20° C., for 21hours. All liquid was removed and the pelleted RNA was resuspended in300 μl 0.3M sodium acetate pH 5.2, then precipitated with 750 μl EtOH at-20° C. The precipitate was resuspended in H₂ 0, then treated withProteinase K to remove any RNAses. After a phenol/chloroform extraction,the RNA was re-precipitated, resuspended in H₂ O and the OD of thesample at 260 nm was measured.

The RNAs were electrophoresed in 1% formaldehyde agarose gels, preparedwith diethyl pyrocarbonate (DEPC) treated solutions. Ten μg of eachtotal RNA sample was loaded per lane. RNA was electrophoresed at 30 Vfor approximately 18 hours with continuous circulation of buffersaccomplished with a peristaltic pump. Each resulting gel was soaked twotimes in 20× SSPE for 20 minutes each at room temperature. Transfer ofRNA to Hybond-C membranes (Amersham Corp., Arlington Heights, Ill.) wasaccomplished by capillary action overnight in 20× SSPE. Using aStratagene stratalinker, RNA was stably crosslinked to each membrane byexposure to ultraviolet light.

To generate ICAM-1 DNA probes, 100-200 ng template DNA (a 1.8 kb Xba/Kpnfragment incorporating the entire ICAM-1 coding sequence) was mixed withH₂ O and random hexamer, boiled for 5 minutes, and then incubated 5minutes on ice. To the template DNA were added: ³² P-dCTP and ³² P-dTTP,10⁻⁴ M dGTP/dATP, 10×Klenow Buffer (Boehringer Mannheim Biochemicals,Indianapolis, Ind.) and Klenow enzyme, and the mixture was left at roomtemperature for 1 hour. Samples were passed over a Quickspin G25 DNAcolumn (Boehringer) to separate incorporated from unincorporated label.

To generate ICAM-R DNA probes, 200 pg of DNA template (a 1.4 kb fragmentof clone pVZ-147 truncated to remove the poly-A tail) was amplified byPCR primed with oligonucleotides complimentary to the 5' and 3'extremities of domain 1. ³² P-dCTP was added to the reaction mixture.Samples were held at 94° C. for 4 minutes then run through 30 cycles ofthe temperature step sequence (94° C., 1 minute; 50° C., 2 minutes; 72°C., 4 minutes) Samples were then run over a Quickspin column andincorporation of label was assessed by scintillation counting of 1 μlaliquots.

The DNA probes were denatured with 5M NaOH, then neutralized with 1MTris. The Hybond-C membranes were prehybridized at 50° C. for 30 minutesin a 50% formamide pre-hybridization mix. Probe was added to eachmembrane to a concentration of 1×10⁶ cpm/ml hybridization mix (50%formamide, 5×Denhardt's solution, 5×SSPE, 1% SDS), and the membraneswere incubated overnight at 42° C. Each membrane was then washed 5 timesin 2× SSPE/0.1% SDS at room temperature for 10 minutes each wash. One 10minute wash was done at 50° C. in 0.5× SSPE/0.1% SDS, with an additionalrinse in 2× SSPE. Hybridization with the major RNA transcript wasquantitated using a Molecular Dynamics (Sunnyvale, Calif.) Model 400APhosphorImager.

Results of the northern blot hybridizations are presented in bar graphform in FIG. 2(A through B). FIG. 2A illustrates specific hybridizationof the ICAM-R probe with RNA extracted from ICAM-R transfectants, butnot with RNA from ICAM-1 transfectants or untransfected L cells.Reciprocally, FIG. 2B indicates hybridization of the ICAM-1 probe withRNA extracted from ICAM-1 transfectants, but not with RNA from ICAM-Rtransfectants or parental L cells.

C. In situ Hybridizations

L cells and L cells transfected as described above with either ICAM-R orICAM-1 cDNAs were hybridized in situ with radiolabelled single-strandedRNA probes derived from ICAM-R or ICAM-1. Single-stranded RNA probeswere generated from DNA templates corresponding to the first (i.e.,N-terminal) immunoglobulin-like domain of ICAM-R or ICAM-1 by in vitroRNA transcription incorporating ³⁵ S-UTP. Probes were chemicallyhydrolyzed to approximately 200 bp.

Transfected and untransfected L cells were layered onto Vectabond(Vector Laboratories, Inc., Burlingame, Calif.) coated slides and storedat -70° C. Prior to use, slides were removed from -70° C. and placed at55° C. for 5 minutes. Sections were then fixed in 4% paraformaldehydefor 20 minutes at 4° C., dehydrated in 70-95-100% EtOH for 10 minutes atroom temperature, and allowed to air dry for 30 minutes. Sections weredenatured for 2 minutes at 70° C. in 70% formamide/2× SSC, rinsed in 2×SSC dehydrated and then air dried for 30 minutes. Prehybridization for 2hours at 42° C. with a mixture containing 50% formamide, 0.3M NaCl, 20mM Tris pH 8.0, 10% dextran sulfate, 1× Denhardt's solution, 100 mMdithiothreitol (DTT) and 5 mM EDTA was performed. Hybridization wascarried out overnight (12-16 hours) at 50° C. in the same mixtureadditionally containing either ³⁵ S-labelled ICAM-1 or ³⁵ S-labelledICAM-R RNA probes (6×10⁵ cpm/section). After hybridization, sectionswere washed for 1 hour at room temperature in 4× SSC/10 mM DTT, then for40 minutes at 60° C. in 50% formamide/1× SSC/10 mM DTT, 30 minutes atroom temperature in 2× SSC, and 30 minutes at room temperature in 0.1×SSC. The sections were alcohol dehydrated, air dried for 30 minutes,developed (after storage at 4° C. in complete darkness) andcounterstained with hematoxylin/eosin.

Photomicrographs of the in situ hybridizations are set out in FIG. 3(Athrough F) wherein photomicrograph 3A is of parental L cells probed withICAM-R RNA; 3B is of ICAM-R transfected L cells probed with ICAM-R RNA;3C is of ICAM-1 transfected L cells probed with ICAM-R RNA; 3D is ofparental L cells probed with ICAM-1 RNA; 3E is of ICAM-R transfected Lcells probed with ICAM-1 RNA; and 3F is of ICAM-1 transfected L cellsprobed with ICAM-1 RNA. The photomicrographs demonstrate specifichybridization of each RNA probe only with L cells transfected with ahomologous cDNA.

EXAMPLE 7

Experiments testing the adhesion of leukocytes to transfected L cellsexpressing ICAM-R on their surface or to soluble ICAM-R (Example 10)indicate that ICAM-R is a ligand/receptor for an adhesion molecule ormolecules on leukocytes.

A. CD18-Dependent Cell Adhesion

SKW3 cells (T lymphoblastoid cells) were pretreated with phorbol esterto activate LFA-1-dependent adhesion as described in Dustin et al.,Nature, 341: 619-624 (1989) and were assayed for binding to ICAM-R andICAM-1 transfectants.

Untransfected L cells or L cells transfected with either ICAM-R orICAM-1 (see Example 7) were seeded in 24-well tissue culture plates(3×10⁵ cells per well) 24-48 hours prior to the adhesion assay. SKW3cells were washed in serum-free RPMI (Gibco, Canada), labelled withCalcein-AM (Molecular Probes Inc., Eugene, Oreg.), and stimulated with10 ng/ml phorbol myristylacetate (PMA) for 20 minutes at 37° C. Selectedstimulated SKW3 cells were then pretreated with anti-CD 18 (TS1/18, ATCCHB203), anti-CD 11 a (TS1/22, ATCC HB202) hybridoma supernatant orcontrol anti-CD2 (ATCC HB195) purified monoclonal antibody for 30minutes at room temperature before incubation with adherent, transfectedL cells. Antibody-treated and non-antibody-treated, calcein-labelledSKW-3 cells were added (5×10⁵ cells per well) to confluent monolayers ofICAM-R or ICAM-1 transfectants and incubated for 30 minutes at 37° C. inRPMI/1% fetal calf serum (FCS, Hyclone Laboratories Inc., Logan, UT)Unbound cells were aspirated and wells were filled with RPMI-FCS. Plateswere sealed, centrifuged in an inverted position at 200 rpm for 4minutes and aspirated. The plates were then washed with RPMI-FCS andscanned with an automatic fluorescence reader.

Adhesion of stimulated SKW3 cells to both the ICAM-R and the ICAM-1transfectants was inhibited by monoclonal antibodies against either theα(CD11a) or β(CD18) chains of LFA-1 indicating that ICAM-R mayparticipate in intercellular adhesion events involving a β2 integrinpathway. Intracellular adhesion was unaffected by the control anti-CD2reagent.

B. CD18-Independent Cell Adhesion

CD18 negative lymphoblastoid cells from patients with leukocyte adhesiondeficiency (LAD) bind to soluble ICAM-R described in Example 10. (SeeFIG. 4A wherein the experimental control was binding of cells to platescoated with 1% BSA.) In addition, the majority (80-90%) of binding ofthe Jurkat T lymphoblastoid cell line to ICAM-R is not inhibited byanti-CD18 monoclonal antibody 60.3 described in Beatty et al., J.Immunol., 131: 2913-2918 (1983)! or anti-CD11a monoclonal antibody(TS1/22) (FIG. 4B). These results suggest that binding of ICAM-R tothese cell lines is CD18-independent and that LAD and Jurkat cellsexpress a counterreceptor for ICAM-R that is not a β₂ integrin.

EXAMPLE 8

Human sequence ICAM-R peptides were used to inhibit SKW3 and Jurkat cellbinding to ICAM-R. The former type of adhesion is CD18-dependent whilethe latter is largely CD18-independent.

Based on amino acid sequence alignment with known β₁ integrin bindingdomains in fibronectin and based on epitope mapping of anti-ICAM-Rmonoclonal antibodies that block cell adhesion (see Table 11 in Example21), ICAM-R peptides corresponding to potential integrin binding siteswere synthesized by Macromolecular Resources (Colorado State University,Fort Collins, Colo.). Four ICAM-R sequences which lie between or at theborder of predicted beta strands in domains 1 and 3 of were chosen.Similar but not identical β-strand predictions for ICAM-10 are set outin Staunton et al., Cell, 61: 243-254 (1990). Inhibition was assayedusing a system involving cell adhesion to soluble ICAM-R coated plastic.Calcein-labeled cells (see Example 7 Section A above) were incubatedwith peptide at 1-2 mg/ml for 20 minutes at 25° C. and the cells weretransferred to wells of a 96-well plate previously coated with solubleICAM-R (see Example 10) and containing 10 μg/ml final concentrationphorbol 12-myristate 13-acetate (PMA). After 50 minutes, the plate wasinverted in PBS for 10 minutes to remove unbound cells. Bound cells werequantitated using a fluorescence concentration analyzer.

The results of the assay are presented below in Table 2 whereinnumbering of peptide residues of ICAM-R corresponds to SEQ ID NO: 1while numbering of peptide residues of ICAM-1 corresponds to the ICAM-1amino acid sequence presented in Staunton et al., supra, and wherein theabbreviation "ND" stands for "not

                  TABLE 2                                                         ______________________________________                                                                % Inhibition                                                                            % Inhibition                                               Peptide  CD18-Dependent                                                                          CD18-Independent                            Protein                                                                              Domain  Residues Binding (SKW3)                                                                          Binding (Jurkat)                            ______________________________________                                        ICAM-R 1       32-38    0%        10%                                                1       72-76    26%       17%                                                3       230-234  0%        36%                                                3       271-276  0%        11%                                         ICAM-1 1       29-35    ND        ND                                                 1       70-74    0%         9%                                                3       228-232  ND        22%                                                3       268-274  ND        ND                                          ______________________________________                                    

ICAM-R peptide sequences from domain 3 inhibited binding of Jurkat cellsto ICAM-R but not binding of SKW3 cells to ICAM-R. Domain 3 peptideswere two-fold more efficient than domain 1 peptide sequences ininhibiting Jurkat cell binding, suggesting that Jurkat binding to ICAM-Rmay preferentially involve ICAM-R domain 3. The ICAM-R domain 1 peptide(NGSQI) corresponding to residues 72-76 of SEQ ID NO: 1 inhibited SKW3binding to ICAM-R by 26%. The corresponding ICAM-1 peptide (DGQST, SEQID NO: 25) did not inhibit binding. In contrast, the ICAM-R domain 3peptide (GDQML) corresponding to amino acids 230-234 of SEQ ID NO: 1demonstrated the best inhibition (36%) of Jurkat binding to ICAM-R. Thecorresponding ICAM-1 peptide (GDQRL, SEQ ID NO: 26) inhibited Jurkatbinding by 22%.

The tri-peptide RGD is a recognition sequence common to extracellularmatrix components (e.g., fibronectin and vitronectin) that are ligandsof the beta-1 integrins. Cyclizing RGD-containing peptides has resultedin a ten-fold increase in efficiency of blocking integrin binding tovitronectin Pierschbacher and Ruoslahti, J. Biol. Chem., 262(36):17294-17298 (1987)!. ICAM-R peptide sequences corresponding to domain 1residues 72-77 and domain 3 residues 230-234 are being cyclized usingbromoacetic acid preparative to testing in the assay outlined above.

EXAMPLE 9

A soluble variant of human ICAM-R was constructed and expressed asfollows.

A. Construction of the Expression Vector Encoding Soluble ICAM-R

The human cDNA for ICAM-R was altered by standard procedures ofsite-directed mutagenesis see, e.g., Kunkel et al., Proc. Natl. Acad.Sci. USA, 82: 488-492 (1985)! in order to truncate the protein codingsequence at the predicted junction (amino acid 457) of its extracellularand transmembrane domains as determined by a computer algorithm thatpredicts hydropathy Kyte et al., J. Mol. Biol., 157: 105-132 (1982)!.The DNA sequence of ICAM-R was cut from pVZ147 (Example 4) withrestriction enzymes SalI and NotI. The resulting fragment included thecomplete ICAM-R coding sequences beginning at the 5' end of the codingstrand and also included at the 3' end a short segment of the multiplecloning sites. This fragment was subcloned into the M13 BM21 vector(Boehringer) linearized with SalI and NotI resulting in a moleculecalled M13 BM21ICAM-R.

A mutagenizing oligonucleotide was synthesized with the sequence below.

ICAM-Rt1(SEQ ID NO: 27)

CTGCCCCTGAATCACCCTCGA

The oligonucleotide changes the phenylalanine at position 457 of ICAM-Rto a stop codon. The oligonucleotide was utilized as described in Kunkelet al., supra, to generate from M13 BM21ICAM-R six M13 phage isolatesencoding a stop codon at position 457. An isolate designatedBM21ICAM-Rt1 was chosen for further study.

This single strand template was converted to a double strand DNAmolecule by primer extension using Klenow DNA polymerase as follows. Tenμg of purified single strand M13 BM21ICAM-Rt1 DNA was annealed to 50 ngLac Z universal -20 primer (GTAAAACGACGGCCAGT, SEQ ID NO: 28) in 1×Klenow DNA polymerase buffer (10 mM Tris-Cl pH 7.5, 5 mM MgCl₂, 7.5 mMdithiothreitol) by incubating the mix at 65° C. for 5 minutes and then25° C. for 5 minutes. The following mixture was then added to theannealing reaction: 33 μM final concentration dATP, dGTP, dCTP, dTTP; 4units of Klenow DNA polymerase (Boehringer), and 1× Klenow buffer. Theprimer extension reaction was allowed to incubate at 37° C. for 45minutes prior to being stopped by a single phenol/chloroform (1:1)extraction and ethanol precipitation. A portion of the cDNA insert wasreleased from the M13 BM21ICAM-Rt1 phage by restriction digest usingrestriction enzymes EcoRV and NcoI. The fragment of DNA releasedcontained the complete coding sequence for the truncated ICAM-R protein,the 3' untranslated region and a small segment of polylinker sequencefrom the M13 BM21 phage. After agarose gel purification the fragment wasligated to linearized vector Bluebac III (Invitrogen Corp., San Diego,Calif.), a transfer vector containing genomic baculovirus sequences forhomologous recombination that flank the ETL promoter driving expressionof the E. coli beta-galactosidase gene and the polyhedron promoterdriving expression of the gene of interest, in this case ICAM-Rt1.

The Bluebac III vector had been prepared in the following way prior toligation. Three μg of supercoiled plasmid DNA was digested with 20 unitsHinDIII endonuclease (Boehringer). After a phenol/chloroform extractionand ethanol precipitation the DNA pellet was resuspended in 1× KlenowDNA polymerase buffer; 33 μM final concentration dATP, dGTP, dCTP, dTTP;2 units of Klenow DNA polymerase (Boehringer) and incubated at 37° C.for 60 minutes to fill in the termini of the molecule. The fill-inreaction was terminated by phenol/chloroform extraction andprecipitation with ethanol. The blunt-ended DNA was resuspended in 1×NcoI buffer, 20 units of NcoI endonuclease were added and incubated at37° C. for 60 minutes.

A portion of the ligation reaction of the ICAM-Rt1 insert and linearizedplasmid was used to transform electro-competent XL-1 E. coli(Stratagene) and individual colonies were selected on LB platessupplemented with 60 μg/ml carbenicillin. Twelve individual isolateswere analyzed by digestion of mini-prep DNA using PstI or EcoRI fordiagnostic purposes. One isolate that exhibited the expected bandpattern was designated pBBIII.ICAM-Rt1.

B. Expression of Soluble Human ICAM-R

Sf-9 cells (Invitrogen) to be transfected or infected with pBBIIIICAM-Rt1 DNA were maintained in spinner flasks in TNM-FH Grace's medium(Gibco, Grand Island, N.Y.) supplemented with 10% heat inactivated fetalbovine serum and gentamicin at 10 μg/ml! at 27° C. in a forced draftincubator. Spinner flask impellers were rotated at 60 rpm on aninsulated five place stir plate. Log phase Sf-9 cells (1.5-2.5×10⁶ /ml)with greater than 90% viability were routinely subcultured twice weekly.

Sf-9 cells at log growth phase were plated (2×10⁶ cells/60 mm dish) inTNM-FH medium and allowed to attach for 1 hour at 27° C. After this timethe following mixture was made up in a sterile polystyrene tube andincubated at room temperature for 15 minutes: 1 ml TMN-FH medium, 1 μglinear Autographa californica nuclear polyhidrosis virus (AcNPV,baculovirus) genomic DNA (Invitrogen), 3 μg of pBBIII.ICAM-Rt1 DNA and20 μl of a stock cationic liposome solution (Invitrogen). Two otherindependent mixtures were made up with or without pBluebac IIIsubstituted for pBBIII.ICAM-Rt1 DNA as controls. The media was removedfrom the seeded plates, replaced with 2 ml of Grace's medium and allowedto incubate for 2 minutes. All media was removed from the plates and theDNA/liposome mixtures were added dropwise on the cells of individualplates. One plate received TNM-FH medium alone as a mock transfectioncontrol. The plates were then incubated at 27° C. for 4 hours withoccasional rocking. Following this incubation, 1 ml of TNM-FH medium wasadded to the plates. After further incubation for 48 hours, thetransfection media containing virus was removed and these viral stockswere used to infect plates of Sf-9 cells for plaque identification.

Sf-9 cells were seeded at 2×10⁶ cells/60 mm dish in TNM-FH medium andallowed to attach for approximately 1 hour at 27° C. The media wasremoved. Several 10-fold serial dilutions were made from each viralstock and 1 μl of each dilution was added to a single dish of adherentSf-9 cells and incubated for 1 hour at 27° C. Following removal of thevirus inoculum, each dish of cells was overlayed with 3 ml of a mixtureof TNM-FH medium, 0.625% low melting point agarose (BRL, Gaithersburg,Md.) and 300 μg/ml halogenated idolyl-beta-D-galactosidase (Bluo-gal,BRL) that had been previously equilibrated to about 30° C. and allowedto solidify at room temperature for 1 hour. The plates were thenincubated until blue color developed (typically 4-5 days). Twenty-fourplaques of recombinant viruses (identified due to their expression ofbeta-galactosidase and conversion of the chromogenic substrate, Bluo-galto a blue precipitate in infected cells) were transferred to individualwells of a 24-well cell culture plate that had been seeded with 1 ml ofSf-9 cells (2×10⁵ / ml) in TNM-FH. After 5 days at 27° C. the media washarvested, microfuged at 1,000 rpm for 5 minutes at 4° C. and theresulting supernatant was transferred to a fresh tube. These stocks weredesignated as BacR.P1 stocks with their respective isolate number.

BacR.P1 stocks were assayed for the production of ICAM-R by an antigencapture (ELISA) assay. Anti-ICAM-R monoclonal antibody ICR-4.2 (seeExample 12) was biotinylated as follows. A tenth volume of 1M NaCO₃ wasadded to monoclonal antibody ICR-4.2 at 1 mg/ml. NHS-biotin (SigmaChemical Co., St. Louis, Mo.) was dissolved into dimethyl sulfoxide(DMSO, Mallinckrodt, Paris, Ky.) at 1 mg/ml. One hundred eighty μlbiotin solution was added to each 1 mg antibody and rotated at 4° C.overnight. The biotinylation reaction was terminated by dialysis againstPBS for 16 hours with 3 changes at 4° C. For the assay of BacR.P1stocks, each well of a ninety-six well plate was coated with monoclonalantibody ICR-1.1 (50 μl at 10 μg/ml) for either 2 hours at 37° C. or 16hours at 4° C. The coating was then aspirated and the wells were rinsed2 times with PBS. Wells were blocked with 200 μl of 1% BSA in PBS for 30minutes at 37° C. Two ten-fold serial dilutions of BacR.P1 stocks weremade in PBS. Fifty μl from the BacR.P1 stocks (neat) or the dilutionswere added to the wells and incubated for 30 minutes at 37° C. After 2washes with PBS, 50 μl for a 1:250 dilution of biotinylated ICR-4.2 in1% BSA/PBS was added to the wells and incubated for 30 minutes at 37° C.After 3 washes with PBS, 50 μl/well of horseradish peroxidase conjugatedto streptavidin (Zymed Laboratories Inc., San Francisco, Calif.) dilutedin 1% BSA/PBS to 1:4000 was added and incubated for 30 minutes at 37° C.After 2 washes with PBS, 200 μl/well substrate buffer with ABTS (Zymed)was added and incubated at room temperature until a color reactiondeveloped. The plate was read in an automated plate reader at awavelength of 410 nm.

Four of the highest expressors of soluble ICAM-R as determined by theabove antigen capture assay were chosen for plaque purification andBacR.P1 stocks of those isolates were diluted by 10-fold serialdilutions and plated with an agar overlay. A single blue plaque from thehighest dilution was isolated and placed in 1 ml of TNM-FH medium,vortexed vigorously and serially diluted for one more round of plaqueisolation. A final plaque isolate was chosen that was clear of allwildtype baculovirus and removed to a T-25 flask that has been seededwith 2×10⁶ Sf-9 cells in TNM-FH media. After 5 days incubation at 27°C., the media was harvested by centrifugation at 1200 rpm for 5 minutesand 4 ml of the supernatant (designated BAC-R.P2 stock) was transferredto a 1 liter spinner flask containing 500 ml of TNM-FH seeded with 2×10⁶cells/ml. After another 5 days incubation at 27° C., the infection mediawas harvested by centrifugation at 1000 rpm for 5 minutes. Thesupernatant was stored at 4° C. and was designated BAC-R.P3 stock. TheBAC-R.P3 stock was titered by plating aliquots of ten fold serialdilutions onto adherent Sf-9 cells and overlaying with 0.625% agarose inTNM-FH supplemented with 300 μg/ml Bluo-gal (BRL). After 4 daysincubation at 27° C., the number of plaques was counted and a titerdetermined.

Infections for expression of soluble ICAM-R protein were carried out in3 liter flasks containing 1.5 L of EX/Cell 401 medium (JRH Biosciences,Lenexa, Kans.). Sf-9 cells dividing at log phase (2×10⁶ /ml) wereinfected at a multiplicity of infection (moi) of 5 with BAC-R.P3 virusstock. After 4 days, the media was harvested and was separated from thecells by centrifugation. Soluble ICAM-R protein was purified from theinsect cell media as follows. Four ml 1M Tris-Cl pH 7.5 was added toeach 200 ml of insect cell supernatant and was pumped at about 35ml/hour at 4° C. onto an approximately 3.5 ml column of Lentil LectinSepharose (Pharmacia, Uppsala, Sweden) previously equilibrated with 20mM Tris-Cl pH 7.5/0.1M NaCl (equilibration buffer). After loading, thecolumn was washed with 25 ml equilibration buffer. The column was theneluted with 11 ml equilibration buffer containing 0.2M methylα-D-mannopyranoside. The eluate contained soluble human ICAM-R(shICAM-R).

C. Binding of shICAM-R to Activated Lymphocytes

The partially purified shICAM-R protein was assayed for binding to SKW3cells that were pretreated with phorbol ester as described in Example 7to activate LFA-1-dependent adhesion. The ICAM-R protein was coated onto96-well Immulon 4 (Dynatech) plates after adjusting the lectin eluate to25 mM carbonate pH 9.6 and incubated overnight at 4° C. The plates werewashed two times with PBS, blocked for 30 minutes at 37° C. with 200ul/well PBS, 1% BSA, and washed again with PBS before adding cells. SKW3cells were washed in serum-free RPMI (Gibco), labelled with Calcein-AM(Molecular Probes), and stimulated with PMA. Cells were then added tothe plates and incubated for 1 hour at 37° C. The plates were invertedin prewarmed PBS, 1% BSA and were incubated for 30 minutes. The plateswere then removed and half of the contents of each well was aspriated.The plates were then scanned with a fluorescence microscope and anautomated fluorescence reader. The results of the assay demonstratedadhesion of phorbol ester-activated lymphocytes to the plate boundshICAM-R protein.

D. Assays Utilizing shICAM-R

In vitro assays for identifying antibodies or other compounds whichmodulate the activity of ICAM-R may be developed that utilize shICAM-R.For example, such an assay may involve immobilizing ICAM-R or a naturalligand to which ICAM-R binds, detectably labelling the nonimmobilizedbinding partner, incubating the binding partners together anddetermining the effect of a test compound on the amount of label boundwherein a reduction in the label bound in the presence of the testcompound compared to the amount of label bound in the absence of thetest compound indicates that the test agent is an inhibitor of ICAM-Rbinding. Functional β₂ leukointegrins that may be utilized in suchassays are described in Dustin et al., CSH Symp. Qual., 54: 753-765(1989).

The following preliminary experiment shows that purified shICAM-R can bebound to polystyrene beads and retain the ability to bind to purifiedleukointegrins coated on a plastic surface, thus providing the basis fordevelopment of an assay to identify modulators of ICAM-R binding.Purified shICAM-R was used to coat 6 μm fluorescent polystyrene beads(Polysciences, Inc., Warrington, Pa.) overnight according to themanufacturer's instructions and then the beads were blocked with BSA.Replicate wells of a 96-well plate were coated with a diluted aliquot ofpurified LFA-1 (CD18/CD11a), Mac-1 (CD18/CD11b) or Gp 150,95(CD18/CD11c). After blocking the wells with BSA, the plates wereincubated in buffer alone or buffer including anti-CD18 antibody (60.3).The ICAM-R-coated beads were aliquoted into the well and incubated forone hour at room temperature followed by inversion in a tank of PBS-D toremove unbound beads from the wells. Fluorescence remaining in the wellswas detected using a Cytofluor 2300 (Millipore, Inc., Bedford, Mass.).In parallel experiments, leukointegrin preparations of LFA-1 or Mac-1were coated on the fluorescent polystyrene beads and ICAM-R wasimmobilized.

Specific modulators of binding between ICAM-R and its binding partnersmay also be identified by scintillation proximity assay techniques asgenerally described in U.S. Pat. No. 4,271,139; Hart et al., Mol.Immunol., 12:265-267 (1979), and Hart et al., J. Nuc. Med., 20:1062-1065(1979), each of which is incorporated herein by reference, may also beutilized to identify modulators.

Briefly, one member of the ICAM-R/ligand pair is bound to a solidsupport. A fluorescent agent is also bound to the support.Alternatively, the fluorescent agent may be integrated into the solidsupport as described in U.S. Pat. No. 4,568,649, incorporated herein byreference. The non-support bound member of the ICAM-R/ligand pair islabelled with a radioactive compound that emits radiation capable ofexciting the fluorescent agent. When, for example, ICAM-R binds theradiolabeled ligand, the label is brought sufficiently close to thesupport-bound fluorescer to excite the fluorescence and cause emissionof light. When not bound, the label is generally too distant from thesolid support to excite the fluorescent agent, and light emissions arelow. The emitted light is measured and correlated with binding betweenICAM-R and the labelled ligand. Addition of a putative modulator to thesample will decrease the fluorescent emission by keeping the radioactivelabel from being captured in the proximity of the solid support.Therefore, binding inhibitors may be identified by their effect onfluorescent emissions from the samples. Potential ligands to ICAM-R mayalso be identified by similar assays in which no modulator is included.

EXAMPLE 10

To rapidly screen for the functional consequences (i.e.,counter-receptor binding) of point mutations in ICAM-R extracellularimmunoglobulin-like domains, a system was employed from which shICAM-Rmolecules having point mutations can be expressed and purified. Thesystem relies on the specific binding properties of a poly-histidinyltract fused to the amino or carboxyl terminus of a given protein Hochuliet al., Bio/Technology, 6: 1321-1325 (1988)!. The utility of the systemin the purification of proteins under native conditions has beendemonstrated Janknecht et al., Proc. Natl. Acad. Sci., USA, 88:8972-8976 (1991)!.

Plasmids pCS57.1 and pCS65.10 both are pcDNAlamp (Invitrogen) with thefull length human ICAM-R cDNA inserted between EcoRV and XhoI sites, butpCS65.10 includes point mutations that encode Ala₃₇ and Ser₃₈ ratherthan the wild type Glu₃₇ and Thr₃₈, respectively! were used for theinitial studies. These DNAs were digested with SacI and EcoRI to releasethe entire extracellular domain of ICAM-R (amino acids -29 to +454) andthe fragments were gel isolated.

Two complimentary oligonucleotides were synthesized that encoded wildtype residues Ser₄₅₄ and Ser₄₅₅, and introduced a Gly₄₅₆, Pro₄₅₇ andGly₄₅₈ to encourage an alpha helical turn followed by a stretch of sixhistidine residues and a translational terminator codon. The sequencesof the oligonucleotides were:

SEQ ID NO: 29

CAGGTCCCGGTCATCATCATCATCATCATTAAT

SEQ ID NO: 30

TAGATTAATGATGATGATGATGATGACCGGGACCTGAGCT

The oligonucleotides which contain a SacI site and an XbaI site at theends were ligated to the extracellular domain of ICAM-R and pcDNA1ampcut with EcoRI and XbaI. One set of ligations contained 0.5 upolynucleotide kinase to phosphorylate the 5' ends of the synthetic DNAsthus increasing the efficiency of ligation. A second set of ligationreactions contained pre-phosphorylated oligonucleotides. Colonies werescreened by either miniprep restriction enzyme digestion analysis andPCR with ICAM-R specific oligonucleotide primers or PCR alone. DNAsequence was obtained for several clones. The resulting plasmids weredesignated p57.1wtHis6 and p65.10E37T His6.

COS cells were seeded in 10 cm dishes and grown to about 50% confluencyat which time they were transiently transfected by the DEAE-dextranmethod in serum free DMEM using 10 ug of purified plasmid DNA per dishor mock transfected. After a brief DMSO shock, the cells were incubatedin DMEM supplemented with fetal bovine serum. After 24 hours, the mediumwas replaced and the cells allowed to reach confluency over the courseof the next four days. The final medium harvest was removed from thecell monolayer and spun at 1000 rpm to remove cells and stored at 4° C.until ready for column chromatography.

Ni⁺⁺ -nitrilotriacetic acid (Ni⁺⁺ -NTA) agarose affinity columnchromatography was performed essentially as described in Janknecht etal., supra, except that the purification was from medium rather thanfrom lysed cells. To the medium was added an equal volume of buffer A(830 mM NaCl, 34% glycerol, 1.6 mM imidazole) and the mixture wasclarified by centrifugation at 10,000×g for 10 minutes at 4° C. One mlof an Ni⁺⁺ -NTA agarose bead suspension (50%) (Qiagen) per 16 mls ofbuffered medium sample was preequilibrated in 3.3 ml of 0.5× buffer A bygentle rocking at 25° C. for 30 minutes. The beads were then spun to apellet at 600 rpm and most of the supernatant was removed. The beadswere resuspended to a total volume of 3 ml in fresh 0.5× buffer A and 1ml dispensed to each clarified and buffered medium sample. The remainderof the prep was carried out at 4° C. After 60 minutes of constantagitation each medium sample was passed through a disposable 10 mlpolypropylene column (Biorad) to pack the beads and the flow throughcollected. The beads were then washed with 9 column volumes (4.5 mls) ofbuffer D (10 mM HEPES pH 7.9, 5 mM MgCl₂, 0.1 mM EDTA, 50 mM NaCl, 1 mMdithiothreitol, 17% glycerol) supplemented with 0.8 mM imidazole. Thebeads were then washed twice with 9 column volumes of buffer Dsupplemented with 8 mM imidazole, twice with 5 column volumes of bufferD supplemented with 40 mM imidazole and twice with 5 column volumes ofbuffer D supplemented with 80 mM imidazole.

Two hundred ul of each fraction were assayed for ICAM-R immunoreactivityby enzyme linked immunofiltration assay (ELIFA) in a 96-well format asdescribed by the manufacturer (Pierce). Purified monoclonal antibodyICR-4.2 (5 ug/ml) (see Example 11) was used as the primary detectionagent and a purified goat anti-mouse horseradish peroxidase conjugate(Boehringer Mannheim Biochemicals) (1:500) was used as the secondaryantibody. The assay was developed with the soluble substrate ABTS(Zymed) as recommended by the supplier and read using a Dynatech platereader with a 410 nm test filter. The results showed that ICAM-Rimmunoreactivity was predominantly found in the first 40 mM imidazolewash.

Peak fractions from wtHis6, E37His6 and mock transfectants wereconcentrated about 6.5 fold using Centricon 30 (Amicon) centrifugationunits. The resultant concentrates were adjusted to equal vols. (0.34 ml)using PBS-D. Control soluble ICAM-R (15 ug/ml) (Example 9) in carbonatebuffer pH 9.6 or in buffer D with 40 mM imidazole were made up. Fifty ulof a protein solution was aliquoted per well of a 96-well plate (Immulon4, Dynatech) to coat the wells which were then assayed for binding ofSKW3 cells as described in Example 9 using untreated, PMA-treated andanti-CD18 monoclonal antibody (60.3) treated cells.

Preliminary results indicate that wild type histidine tagged protein(wtHis6) functions as an adhesive ligand for SKW3 cells.

EXAMPLE 11

Monoclonal antibodies specific for ICAM-R were generated from the fusionof NS-1 myeloma cells with spleen cells of Balb/c mice immunized withhuman cell lines that express ICAM-R. Monoclonal antibodies weregenerated from seven different fusions designated fusions 26, 42, 43,46, 56, 63, and 81.

A. Immunization of Mice

For fusion 26, five 6 to 12-week old Balb/c mice (Charles RiverBiotechnical Services, Inc., Wilmington, Mass., IACUC #901103) wereimmunized with HL-60 cells to generate anti-ICAM-R monoclonalantibodies. Two Balb/c mice were bled retro-orbitally for the collectionof pre-immune serum on day 0. On day 2, each animal received a total of6×10⁶ HL-60 cells in 0.5 ml PBS (0.1 ml s.c. and 0.4 ml i.p.). A secondimmunization with 9.5×10⁶ HL-60 cells was administered on day 28 in thesame manner. Immune serum was collected via retro-orbital bleeding onday 35 and tested by FACS (FACS screening is described in detail inSection C below) to determine its reactivity to ICAM-R transfectants.Based on these results, both animals were immunized a third time on day51 with 6.5×10⁶ HL-60 cells and a fusion was performed with spleen cellssterilely removed from one animal (#764) on day 54.

For fusion 42, on day 0 each of five mice was prebled and then immunizedi.p. with 5×10⁶ SKW3 cells in 0.5 ml PBS containing 50 μg adjuvantpeptide (Sigma). The mice were boosted in the same manner on days 21 and42. Ten days after the third injection, the mice were bled and immunesera was tested by FACS. Mouse #843 was given a final boost of SKW3cells on day 64. The spleen was sterilely removed three days later.

For fusion 43, on day 0 each of five mice was prebled and then immunizedi.v. with 5×10⁶ cells from the erythroleukemic cell line K562. Eachmouse was given a daily i.p. injection of 1.5 mg cyclophosphamide in 150μl for the next two days. On day 10, SKW3 cells plus adjuvant peptidewere injected as in Fusion 42. On day 30, mice were given another cycleof K562 cells followed by cyclophosphamide. On day 42 mice were boostedwith SKW3 cells with adjuvant peptide. Mice were bled on day 56 andimmune sera was tested by FACS. Mouse #1021 was given a final boost ofSKW3 cells and adjuvant peptide on day 78. The spleen was sterilelyremoved three days later.

For fusion 46, a mouse (#900) was immunized as described for fusion 42.On day 128, the mouse was given a final boost of approximately 4×10⁶Macaca nemestrina spleen cells. The single cell suspension of monkeyspleen was prepared as described below in the following paragraph. Themonkey cells were pelleted and resuspended in erythrocyte lysis buffer:0.15M NH₄ Cl, 1M KHCO₃, 0.1 mM Na₂ EDTA, pH 7.2-7.4. After lysing theerythrocytes, the splenocytes were washed twice in RPMI and once in PBS.Finally, the cells were resuspended in 400 μl PBS containing 50 μgadjuvant peptide and injected. The mouse spleen was removed sterilelythree days later.

For fusions 56 and 63, mice (#845 and #844) were immunized as describedfor fusion 42, except that no boost of SKW3 cells was given on day 64.Instead, these mice were given additional immunizations of SKW3 cells inPBS with adjuvant peptide on days 158 and 204 and were given i.p.injections of Macaca nemestrina spleen cells in 0.5 ml PBS containing 50μg adjuvant peptide on days 128 and 177. For fusion 56, mouse #845 wasinjected with 2.24 μg soluble ICAM-R (Example 10) in 700 μl PBS, 100 μlwas given i.v. with the remainder given i.p. The spleen was sterilelyremoved four days later. For fusion 63, mouse #844 was immunized on day226 with Macaca nemestrina spleen cells as described for fusion 56 andon day 248 with 50 μg soluble ICAM-R in 100 μl complete Freuds adjuvantgiven s.c. The mouse received a final boost i.v. of 66 μg soluble ICAM-Rin 100 μl PBS. The spleen was removed sterilely four days later.

For Fusion 81 each of 5 mice was prebled on day 0 and then immunizeds.c. with 30 ug of soluble human shICAM-R (Example 9) in 0.2 ml completeFreund's adjuvant. On days 45 and 77, each mouse received 40 μg ofshICAM-R in 0.2 ml incomplete Freund's adjuvant. On day 136 mouse #1264(Fusion 81) was given a final boost i.p. of 0.1 mg of shICAM-R in PBS.The spleen was sterilely removed three days later and a fusion wasperformed as described above.

B. Fusions

Briefly, a single-cell suspension was formed from each mouse spleen bygrinding the spleen between the frosted ends of two glass microscopeslides submerged in serum free RPMI 1640 (Gibco), supplemented with 2 mML-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100μg/ml streptomycin (Gibco). The cell suspension was filtered throughsterile 70 mesh Nitex cell strainer (Becton Dickinson, Parsippany,N.J.), and washed twice by centrifuging at 200 g for 5 minutes andresuspending the pellet in 20 ml serum free RPMI. Thymocytes taken fromthree naive Balb/c mice were prepared in a similar manner.

NS-1 myeloma cells, kept in log phase in RPMI with 11% fetal bovineserum (FBS) or Fetalclone (Hyclone) for three days prior to fusion, werecentrifuged at 200 g for 5 minutes, and the pellet was washed twice asdescribed in the foregoing paragraph. After washing, each cellsuspension was brought to a final volume of 10 ml in serum free RPMI,and 10 μl was diluted 1:100. Twenty μl of each dilution was removed,mixed with 20 μl 0.4% trypan blue stain in 0.85% saline (Gibco), loadedonto a hemacytometer (Baxter Healthcare Corp. Deerfield, Ill.) andcounted.

A sample of 2×10⁸ spleen cells was combined with 4×10⁷ NS-1 cells,centrifuged and the supernatant was aspirated. The cell pellet wasdislodged by tapping the tube and 2 ml of 37° C. PEG 1500 (50% in 75 mMHepes, pH 8.0) (Boehringer) was added with stirring over the course of 1minute, followed by adding 14 ml of serum free RPMI over 7 minutes. Anadditional 16 ml RPMI was added and the cells were centrifuged at 200 gfor 10 minutes. After discarding the supernatant, the pellet wasresuspended in 200 ml RPMI containing 15% FBS or Fetalclone, 100 μMsodium hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine (HAT) (Gibco),25 units/ml IL-6 (Boehringer) and 1.5×10⁶ thymocytes/ml. The suspensionwas dispensed into ten 96-well flat bottom tissue culture plates at 200μl/well. Cells in plates were fed three times typically on 2, 4, and 6days post fusion by aspirating approximately 100 μl from each well withan 18 G needle (Becton Dickinson), and adding 100 μl/well plating mediumdescribed above except containing 10 units/ml IL-6 and lackingthymocytes.

C. Screening

When cell growth reached 60-80% confluency (day 8-10), culturesupernatants were taken from each well of Fusions 26 and 42, pooled bycolumn or row and analyzed by FACS on parental L cells (Fusion 26) orparental CV-1 cells (Fusion 42); (negative control) and on L cells(Fusion 26) or CV-1 cells (Fusion 42) transfected with ICAM-R DNA.Briefly, transfected and nontransfected L cells or CV-1 cells werecollected from culture by EDTA (Versene) treatment and gentle scrapingin order to remove the cells from the plastic tissue culture vessels.Cells were washed two times in Dulbecco's PBS with Ca²⁺ and Mg²⁺, onetime in "FA Buffer" (either D-PBS or RPMI 1640, 1% BSA, 10 mM NaN₃), anddispensed into 96-well round bottomed plates at 1.5-2.0×10⁵ cells/100 μlFA Buffer per well. At this point, the assay was continued at 4° C.Cells were pelleted by centrifugation in a clinical centrifuge at 4° C.The supernatant from each well was carefully suctioned off, the pelletswere broken up by gently tapping all sides of the assay plate. Onehundred μl of hybridoma supernatant pool was added per well using a12-channel pipetman. Each monoclonal antibody-containing supernatantpool was incubated for 1 hour on both parental and transfected cells at4° C. Assay plates were then washed 2 times with FA Buffer as above. Thelast wash was replaced with a 50 μl/well of a 1:100 dilution of aF(ab')₂ fragment of sheep anti-mouse IgG (whole molecule)-FITC conjugate(Sigma) prepared in FA Buffer. Assay plates were incubated at 4° C.protected from light for 45 minutes. The assay plates were then washed 2times with D-PBS containing NaN₃ only (i.e., no BSA) in the same manneras before and the last wash was replaced with 200 μl/well 1%paraformaldehyde in D-PBS. Samples were then transferred to polystyrenetubes with the aid of a multichannel pipet for flow cytometric analysis(FACS) with a Becton Dickinson FACscan analyzer.

Fusions 43 and 46 were screened initially by antibody capture ELISA,testing for the presence of mouse IgG in hybridoma supernatants.Immunlon 4 plates (Dynatech, Cambridge, Mass.) were coated at 4° C. with50 μl/well goat anti-mouse IgA, IgG or IgM (Organon Teknika Corp.,Durham, N.C.) diluted 1:5000 in 50 mM carbonate buffer, pH 9.6. Plateswere washed 3 times with PBS with 0.05% Tween 20 (PBST) and 50 μlculture supernatant was added. After incubation at 37° C. for 30minutes, and washing as above, 50 μl of horseradish peroxidaseconjugated goat anti-mouse IgG(fc) (Jackson ImmunoResearch, West Grove,Pa.) diluted 1:3500 in PBST was added. Plates were incubated as above,washed 4 times with PBST and 100 μl substrate, consisting of 1 mg/mlo-phenylene diamine (Sigma) and 0.1 μl/ml 30% H₂ O₂ in 100 mM Citrate,pH 4.5, was added. The color reaction was stopped in 5 minutes with theaddition of 50 μl of 15% sulfuric acid. A₄₉₀ was read on an automaticplate reader.

Fusions 56 and 63 were screened initially by antigen capture ELISA.Immulon 4 plates (Dynatech) were coated at 4° C. overnight with 100 ng26E3D Fab' (see Section F below) per well, diluted in 50 mM carbonatebuffer. The plates were blocked with 100 μl/well 2% BSA in PBS for 1hour at ambient temperature. After the plates were aspirated, culturesupernatant containing soluble ICAM-R was diluted 1:8 in PBST and addedat 50 μl/well. After 1 hour incubation at ambient temperature, the wellswere washed three times with PBST, hybridoma culture supernatant wasadded at 50 μl/well, and the plates were again incubated as above. Theplates were washed 3 times and 50 μl/well peroxidase conjugated goatanti-mouse IgG diluted 1:3500 in PBST was added. The remainder of theassay was performed as described in the foregoing paragraph.

Fusion 81 was screened by ELISA on COS cells transiently transfectedwith either a domain 1 deleted ICAM-R construct Example 14.C.1! or withan ICAM-2 construct. The transfected cells were paraformaldehyde fixedon 96-well plates, and the remainder of the assay was performed aspreviously described, except no Tween 20 was used. The wells that werepositive for domain 1 deleted ICAM-R but negative for ICAM-2, weretested on Cos cells transiently transfected with domain 1 deleted ICAM-Ror domain 3 deleted ICAM-R by ELISA.

D. Subcloning

Supernatants from individual wells representing the intersection pointsof positive columns and rows (Fusions 26 and 42), individual wellsproducing IgG (Fusions 43 and 46), or individual wells reactive withsoluble ICAM-R (Fusions 56 and 63) were rescreened by FACS the followingday. L cells or L cells transfected with ICAM-R DNA were used forscreening Fusion 26 antibodies and CV-1 cells or CV-1 cells transfectedwith ICAM-R DNA were used for screening antibodies from Fusions 42, 43,46, 56 and 63. Twenty-nine wells (designated 26E3D-1, 26E3E, 26H3G,26H11C-2, 26I8F-2, 26I10E-2, 26I10F, 42C5H, 42D9B, 43H7C, 46D7E, 56D3E,56I4E, 63A10E, 63C3F, 63C11A, 63E9G, 63E12C, 63G3G, 63H6H, 63H9H, 63I1C,63I6G, 63I12F, 63G4D, 63E11D, 63H4C, showed preferential staining of theICAM-R transfectants versus the control cells. Fusion 81 was alsorescreened by FACS. One well from fusion 81, designated 81K2F, waspositive on ICAM-R domain 1 deletion cells but negative on domain 3deletion cells (Example 14). The well was subcloned successively usingRPMI, 15% FBS, 100 μM sodium hypoxanthine, 16 μM thymidine, and 10units/ml IL-6. Subcloning was performed either by doubling dilution orby limiting dilution, by seeding 96 well plates at 0.5-1.0 cells/well.Wells of subclone plates were scored visually after 4 days and thenumber of colonies in the least dense wells were recorded. Selectedwells of each cloning were tested, by FACS or ELISA as described, forreactivity observed in the original fusion well. Activity was retainedin sixteen cell lines which were deposited with the ATCC 26E3D-1 (ATCCHB 11053), 26H11C-2 (HB 11056), 26I8F-2 (HB 11054), 26I10E-2 (ATCC HB11055), 42C5H (ATCC HB 11235), 42D9B (ATCC HB 11236), 43H7C (ATCC HB11221), 46D7E (ATCC HB 11232) and 46I12H (ATCC HB 11231), 63E11D (ATCCHB 11405), 63G4D (ATCC HB 11409), 63H4C (ATCC HB 11408), 63H6H (ATCC HB11407), 63I1C (ATCC HB 11406), 63I6G (ATCC HB 11404), and 81K2F (ATCC HB11692). In the final cloning, positive wells containing single colonieswere expanded in RPMI with 11% FBS. Names assigned to the monoclonalantibodies produced by the hybridomas are presented in Table 4 inExample 12.

E. Characterization

The monoclonal antibodies produced by above hybridomas were isotyped inan ELISA assay. Immulon 4 plates (Dynatech) were coated at 4° C. with 50μl/well goat anti-mouse IgA, IgG or IgM (Organon Teknika) diluted 1:5000in 50 mM carbonate buffer, pH 9.6. Plates were blocked for 30 minutes at37° C. with 1% BSA in PBS, washed 3 times with PBS with 0.05% Tween 20(PBST) and 50 μl culture supernatant (diluted 1:10 in PBST) was added.After incubation and washing as above, 50 μl of horseradish peroxidaseconjugated rabbit anti-mouse IgG₁, G_(2a), G_(2b) or G₃ (Zymed) diluted1:1000 in PBST with 1% normal goat serum was added. Plates wereincubated as above, washed 4 times with PBST and 100 μl substrate,consisting of 1 mg/ml o-phenylene diamine (Sigma) and 0.1 μl/ml 30%hydrogen peroxide in 100 mM Citrate, pH 4.5, was added. The colorreaction was stopped in 5 minutes with the addition of 50 μl of 15%sulfuric acid. A₄₉₀ was read on a plate reader. The isotypes of themonoclonal antibodies are give in Table 11 in Example 21.

FACS analyses of indirect immunofluorescence staining of control cellsand cells transfected with ICAM-R or ICAM-1 DNA using monoclonalantibodies against ICAM-R, ICAM-1 and ICAM-2 were performed. Stainingwas carried out as described for FACS analyses in Example 12C usingeither 0.1 ml hybridoma culture supernatant (anti-ICAM-R) or 1 μg puremonoclonal antibody (anti-ICAM-1 or ICAM-2) per 5×10⁵ cells. Results ofthe analyses are presented as histograms (representing 10⁴ cellsanalyzed) in FIG. 5. Anti-ICAM-R antibodies specifically bound to Lcells transfected with ICAM-R cDNA, but not to parental or ICAM-1transfected L cells. ICAM-R transfectants did not react with antibodiesagainst ICAM-1 (Mab LB2 from Edward Clark, University of Washington) orICAM-2 (IC2/2, Biosource Genetics Corp., Vacaville, Calif.).

FACS analysis of indirect immunofluorescence of Macaca fascicularis,porcine or canine peripheral blood leukocytes was performed using theanti-ICAM-R monoclonal antibodies. Twenty ml of heparinized Macacafascicularis blood or porcine blood was diluted with 280 ml oferythrocyte lysis buffer, incubated 3-5 minutes at room temperature, andcentrifuged at 200 g for 5 minutes. The supernatant was discarded. Thepellet was washed once in cold D-PBS containing 2% fetal bovine serumand the cells were counted by hemacytometer. Twenty ml of heparinizedcanine blood was diluted in two volumes of Waymouth's medium (Gibco)plus 2% nonessential amino acids (NEAA). Each 5 ml of blood solution waslayered over 4 ml of Histopaque (Sigma) and centrifuged at 1000 g for 20minutes at room temperature. Cells were collected from the interface,washed once in Waymouth's medium plus 2% NEAA, and counted as above.Each cell population was stained as described previously in Example 12Cand analyzed by FACS. Anti-ICAM-R antibodies produced by hybridoma celllines 26I10E, 46I12H, 63H4C, 56I4E and 63I12F specifically stainedmonkey PBL while the other antibodies did not. None of the antibodiesspecifically stained canine or porcine PBL. The monoclonal antibodiesproduced by the hybridoma cell lines 63A10E, 63E9G, 63E12C, 63G3G and63H9H were not tested.

F. Purification

Hybridoma culture supernatants containing the anti-ICAM-R monoclonalantibodies listed in Table 11 in Example 21 were adjusted to 1.5Mglycine, 3.0M NaCl, pH 8.9, and put over a 2 ml bed volume protein Acolumn (Sigma). After washing with 1.5M glycine, 3M NaCl, pH 8.9, thecolumn was eluted with 100 mM sodium citrate, pH 4.0. One ml fractionswere collected into 100 μl of 1.5M Tris, pH 8.8. Fractions containingantibody as determined by A₂₈₀ were pooled and dialyzed against PBS.

G. Affinity

Nine of the purified anti-ICAM-R monoclonal antibodies were dilutedserially and assayed in an ELISA format for binding to a fixed amount ofsoluble ICAM-R (Example 9) coated onto plastic. The results of the assayare presented in Table 3 below wherein high affinity binding was definedas 50% maximal binding at a monoclonal antibody concentration of lessthan 1 μg/ml and low affinity binding was defined as 50% maximal bindingat a monoclonal antibody concentration of greater than 1 μg/ml.

                  TABLE 3                                                         ______________________________________                                        Monoclonal Antibody                                                                  Produced By    Affinity                                                ______________________________________                                               26E3D          Low                                                            26H11C         High                                                           26I8F          High                                                           26I10E         Low                                                            42C5H          Low                                                            42D9B          Low                                                            43H7C          Low                                                            46D7E          High                                                           46I12H         Low                                                     ______________________________________                                    

F. Fab' Fragment Production

Fab' fragments were generated from the monoclonal antibodies produced byhybridomas 26E3D, 26I10E, 42D9B, 43H7C and 46D7E by the method describedin Johnstone et al., p. 52 in Blackwell, Immunochemistry in Practice,Oxford Press (1982).

EXAMPLE 12

ICAM-R specific monoclonal antibodies listed in Table 11 in Example 21were tested for their ability to inhibit binding of JY cells (CD18⁺) torecombinant soluble human ICAM-R. Adhesion assays were performed asdescribed in Example 9. Cells were treated with PMA and antibodies werethen added at a final concentration of 10 μg/ml. Data was collected fromtriplicate wells during three independent experiments. Total CD18-dependent binding was determined as the amount of adhesion blocked bya control anti-CD 18 monoclonal antibody 60.3. The percentage of totalCD18-dependent binding that was inhibited by each monoclonal antibody isshown below in Table 4 wherein the names assigned to monoclonalantibodies produced by each hybridoma are given and "ND" indicates theantibody was not tested. The monoclonal antibody names are usedthroughout the following examples instead of hybridoma designations.

                  TABLE 4                                                         ______________________________________                                        Hybridoma                                                                              Monoclonal Antibody                                                                         Inhibition (%)                                                                           Standard Error                              ______________________________________                                        --       60.3          100        20                                          26E3D    ICR-1.1       45         10                                          26H11C   ICR-2.1       5          7                                           26I8F    ICR-3.1       40         9                                           26I10E   ICR-4.2       3          12                                          42C5H    ICR-5.1       25         10                                          42D9B    ICR-6.2       2          5                                           43H7C    ICR-7.1       10         15                                          46D7E    ICR-8.1       75         10                                          46I12H   ICR-9.2       2          10                                          63E11D   ICR-12.1      20         8                                           63G4D    ICR-13.1      15         20                                          63H4C    ICR-14.1      70         13                                          63H6H    ICR-15.1      43         15                                          63I1C    ICR-16.1      46         13                                          63I6G    ICR-17.1      68         15                                          81K2F    ICR-19.3      ND         ND                                          ______________________________________                                    

EXAMPLE 13

Monoclonal antibodies ICR-8.1 and ICR-1.1 were humanized as follows.

A. ICR-8.1 Humanization

1. RNA Isolation

ICR-8.1 hybridoma cells were grown in RPMI 1640 plus 10% FBS to about4×10⁵ cells per ml. 4×10⁷ cells were harvested by centrifugation, washedtwice in ice-cold PBS and lysed in 5 ml RNAStat (Tel-Test B Inc.Friendswood, Tex.). After extraction with chloroform, the RNA wasprecipitated with isopropanol, collected by centrifugation, washed in70% ethanol, dried and dissolved in 600 μl water. The yield wasdetermined spectrophotometrically as 1.4 mg.

2. Isolation of ICR-8.1 V region cDNA

The heavy chain of the ICR-8.1 murine antibody is of the IgG₁ subclass.V_(H) cDNA was reverse transcribed from RNA primed with anoligonucleotide,

CG1FOR (SEQ ID NO: 31)

GGAAGCTTAGACAGATGGGGGTGTCGTTTTG,

which is based on amino acids 114-122 of the murine IgG₁ constant region(Kabat et al., in Sequences of Immunological Interest, U.S. Departmentof Health and Human Services, NIH, 1991). The primer includes a HindIIIsite for directional cloning. The light chain of the murine antibody isof the kappa class. V_(K) cDNA was reverse transcribed from RNA primedwith an oligonucleotide,

LKC-1 (SEQ ID NO: 32)

GCTATCGGATCCACTGGATGGTGGGAAGATGGA,

which is based on amino acids 116-122 of the murine kappa constantregion (Kabat et al., supra). The primer includes a BamHI site fordirectional cloning.

cDNA reactions in a volume of 50 μl consisted of 5 μg ICR-8.1 RNA, 50 mMTris HCl pH 8.5, 8 mM MgCl₂, 30 mM KCl, 1 mM DTT, 25 pmol CG1FOR orLKC-1, 250 μM each of dATP, dCTP, dGTP and dTTP and 20u RNase inhibitor(Boehringer Mannheim). Oligonucleotides were annealed to the RNA byheating at 70° C. for 5 minutes and slowly cooling to 42° C. Then, 11uAMV reverse transcriptase (Boehringer Mannheim) was added and incubationat 42° C. continued for 1 hour.

V_(H) and V_(K) cDNAs were amplified using a battery of primers based onthe mature N-terminal regions of known murine V_(H) and V_(K) genes(Kabat et al., supra). For V_(H) these oligonucleotides were:

HFR1-1 (SEQ ID NO: 33)

CGATACGAATTCSADGTRCAGCTKMAGGAGTCRGGA,

HFR1-2 (SEQ ID NO: 34)

CGATACGAATTCSAGGTYCARCTKCARCARYCTGG,

HFR1-3 (SEQ ID NO: 35)

CGATACGAATTCGARGTGAAGCTKSWSGAGWCTGG,

HFR1-4 (SEQ ID NO: 36)

CGATACGAATTCAGGTSMARCTGCAGSAGTCWG, and

HFR1-6 (SEQ ID NO: 37)

CGATACGAATTCSAGGTSMARCTGCAGSARHC.

These primers include an EcoRI restriction site (underlined) fordirectional cloning. For V_(K) the primers were:

LFR1-1 (SEQ ID NO: 38)

CGATACGAATTCSAAAWTGTKCTSACCCAGTCTCCA,

LFR1-2 (SEQ ID NO: 39)

CGATACGAATTCGACATTGTGMTGWCMCARTCTCC,

LFR1-3 (SEQ ID NO: 40)

CGATACGAATTCGATRTTKTGATGACYCARRCTSCA, and

LFR1-4 (SEQ ID NO: 41)

CGATACGAATTCGAYATYSWGATGACMCAGWCTMC.

The N-terminal V region primers were used in concert with the CG1FOR andLKC-1 primers to amplify the V_(H) and V_(K) cDNAs by PCR. The mixturesfor the PCR consisted of 5 μl cDNA, 10 mM Tris HCl pH 8.3, 50 mM KCl,1.5 mM MgCl₂, 250 μM each of dATP, dCTP, dGTP, dTTP, 0.01% (v/v) Tween20, 0.01% (w/v) gelatin, 0.01% (v/v) NP-40, 25 pmol CG1FOR or LKC-1, 25pmol HFR1-1, HFR1-2, HFR1-3, HFR1-4 or HFRH1-6, or LFR-1-1, LFR1-2,LF1-3 or LFR1-4 and 2.5 u Thermalase (IBI, New Haven, Conn.) in areaction volume of 50 μl. Samples were subjected to 25 thermal cycles of94° C., 30 seconds; 50° C., 30 seconds; 72° C., 1 minute. Aliquots wereanalysed by agarose gel electrophoresis. Products of the expected sizewere found for all combinations of V_(H) primers and all combinations ofV_(K) primers except for LKC-1 and LFR1-1.

The V_(H) DNA was cut with EcoRI and Hindll, and the V_(K) DNA cut withEcoRI and BamHI. Both DNA types were cloned into M13 BM21 (BoehringerMannheim) and M13 tg 130 and the DNA sequence of the inserts determined.Full-length functional V region sequences were obtained. By comparisonwith other murine V region sequences the ICR-8.1 murine V_(H) and V_(K)genes were members of murine heavy chain subgroup IIA and murine kappasubgroup II (Kabat et al., supra). In order to determine the authenticN-terminal sequences of both heavy and kappa V regions, PCRs were donewith oligonucleotides based on the known signal sequences of murineheavy chain subgroup IIA and kappa subgroup II. SEQ ID NOs: 42 and 43,and 44 and 45 respectively show the entire DNA and amino acid sequencesof ICR-8.1 murine V_(H) and V_(K).

3. Humanized ICR-8.1 V_(H)

The murine V_(H) sequence was aligned with consensus sequences of humanV_(H) subgroups (Kabat et al., supra) and was found to be mosthomologous to human subgroup I. Therefore, a consensus human subgroup Iwas chosen as the framework for receiving the murine complementaritydetermining regions (CDRs).

The template for humanization was single-stranded DNA encoding aconsensus sequence of human subgroup I containing irrelevant CDRs clonedin M13. The M13 clone was grown in E. coli RZ1032 (dut⁻ ung⁻) so thatits DNA contained uracil instead of thymine. For grafting the CDRsequences onto the human framework region (FR), oligonucleotides weresynthesised, each encoding a CDR and flanked at both ends by nucleotidescorresponding to the human template DNA. The sequences of the mutagenicprimers were:

CDR1 (SEQ ID NO: 46)

GGCCTGTCGCACCCAGAGTATGATGCAGTCAGTGAAGR

TGTATCC,

CDR2 (SEQ ID NO: 47)

TGTGTCCRCGGTAATGGTCACTCTGCCCTTGAATTTCA

GATTATAGGTAGTAGTACCAAAGTAAGGATTAATTTTTCCC

ATCCATTCGAG,

CDR3 (SEQ ID NO: 48)

TCCTTGGCCCCCAGTAGTCCATAGCATCTGGGTAGGCC

TCCTTTCTTGCACAGTAATACACGG

Ten pmol of each oligonucleotide was phosphorylated in 25 μl 100 mM TrisHCl pH 8.0, 10 mM MgCl2, 7 mM DTT, 1 mM ATP and 5 u polynucleotidekinase (Boehringer Mannheim) for 1 hour at 37° C. Primers were annealedto the template in a 20 μl reaction mixture consisting of 0.2 pmoltemplate, 2 pmol each phosphorylated oligonucleotide, 100 mM Tris HClpH, 10 mM MgCl₂ and heating to 90° C. for 10 seconds, followed by rapidcooling to 70° C. and slow cooling to room temperature. To the annealedDNA was added 2 μl 0.1M DTT, 2 μl 0.5 M Tris HCl pH 8.0, 0.1 M MgCl₂, 2μl 0.1 M ATP, 1 μl 6.25 mM each of dATP, dCTP, dGTP, and dTTP, 2.5 u T7DNA polymerase (United States Biochemicals, Cleveland, Ohio), 0.5 u T4DNA ligase (Boehringer Mannheim) and 3.7 μl water. Incubation was for 2hours at 22° C. The DNA was ethanol precipitated, washed, dried anddissolved in 50 μl 60 mM Tris HCl pH 8.0, 1 mM EDTA, 1 mM DTT, 0.1 mg/mlBSA and 1 u uracil-DNA glycosylase (Boehringer Mannheim) and incubatedat 37° C. for 1 hour. Phosphodiester bonds at apyrimidinic sites werecleaved by making the sample 0.2 M NaOH and incubating at 22° C. for 5minutes. The sample was neutralized by the addition of 0.5 vol 7.5 Mammonium acetate and the DNA precipitated with ethanol. The washed anddried DNA was finally dissolved in 20 μl 10 mM Tris HCl, 1 mM EDTA pH8.0. The sample containing mutated DNA was amplified by PCR in areaction mixture containing 2 μl mutant DNA mix, 250 μM each of dATP,dGTP, dCTP and dTTP 10 mM Tris HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl₂,0.01% (v/v) Tween 20, 0.01% (w/v) gelatin, 0.01 (v/v) NP-40, 25 pmol M13universal sequencing primer (GTAAAACGACGGCCAGT, SEQ ID NO: 49), 25 pmolM13 reverse sequencing primer (AACAGCTATGACCATG, SEQ ID NO: 50) and 2.5u Thermalase (IBI). Samples were subjected to 15 thermal cycles of 94°C., 30 seconds; 50° C., 30 seconds; 72° C., 45 seconds.

Because humanized V regions often exhibit lower affinities than theirprogenitor antibodies, the primers used for the mutagenesis had thepotential to encode either threonine or serine at position 28 andalanine or valine at position 71, sites which can play a role in antigenbinding. The DNA was cut with NotI and BamHI and cloned into M13 BM21for sequence determination. An M13 clone (designated M13 HuVHV)containing the fully humanized V region with no spurious mutations andwith Thr28 and Val7l Ala71 ted. A version containing Ala71 was made byPCR overlap and extension Ho et al., Gene, 77: 51-59 (1989)!. In thefirst PCRs the primers were oligonucleotide 42 (ACCATTACCGCGGACACATCCAC,SEQ ID NO: 51) with the M13 universal sequencing primer and mutagenicCDR2 oligonucleotide primer with the M13 reverse sequencing primer.Reaction conditions for the PCR were as in Section A.2 above except that5 pmol of primer 42 and the CDR2 primer and 1 u Thermalase were used andthere were 15 cycles of 94° C., 30 seconds; 40° C., 30 seconds; 72° C.,30 seconds. The two product DNAs were joined in a second PCR whichconsisted of 1 μl DNA from the first PCRs in 50 μl 20 mM Tris HCI pH8.2, 10 mM KCl, 6 mM (NH₄)₂ SO₄, 2 mM MgCl₂, 0.1% (v/v) Triton X-100, 10μg/ml BSA, 250 mM each of dATP, dCTP, dGTP, dTTP 25 pmol each of M13universal and reverse sequencing primers and 1 u Pfu DNA polymerase(Stratagene). The sample was subjected to 15 thermal cycles of 94° C.,30 seconds; 50° C., 30 seconds; 75° C., 30 seconds. The DNA was cut withNotI and BamHI, cloned into M13 BM21 and the required clone containingthe Ala 71 mutation selected by DNA sequencing. This DNA was designatedICR-8.1 HuVH and its sequence and deduced translation product are shownin SEQ ID NOS: 52 and 53. In addition, to reduce as far as possible thepotential immunogenicity of the humanized antibody, a V_(H) region wasmade with only the structural loop residues of murine origin togetherwith CDR residures, which from cystallographic data of other antibodies,indicate a role in antigen-binding. Thus, residues at V_(H) positions60, 61 and 64 were changed to consensus residues of human subgroup I.The mutations were Asn60 to Ala, Leu61 to Gln and Lys64 to Gln. Themutations were introduced by PCR overlap and extension (Ho et al.,supra) using oligonucleotides 51 (TACTACCTATGCTCAGAAATTCCAGGGCAGAG, SEQID NO: 54) and 52 (CTCTGCCCTGGAATTTCTGAGCATAGGTAGTAG, SEQ ID NO: 55),essentially as described for introduction of the Ala71 mutation. Theamplified DNA was cut with NotI and BamHI, cloned into M13 BM21 and aclone containing the desired mutation was identified by DNA sequencing.This DNA and encoded protein were designated ICR-8.1 miHuVH.

Two additional versions based on miHuVH were made both containing theamino acid change Val2 to Ile2 (designated miHuVHI) and one additionallycontaining the amino acid change Ser7 to Thr7 (designated miHuVHIT). ThemiHuVHI version was made using the miHuVH DNA as a template formutagenesis via PCR overlap and extension with oligonucleotides 81(CACAGGTGTCCACTCCCAGATCCAGCTGG, SEQ ID NO: 56) and 82(TGGGAGTGGACACCTGTGGAGAGAAAGGCAAAGTGG, SEQ ID NO: 57).

The miHuVHIT version was similarly made using oligonucleotides 82 and 84(CACAGGTGTCCACTCCCAGATCCAGCTGGTGCAGACTGGGGC, SEQ ID NO: 58).

4. Humanized ICR-8.1 V_(K)

The murine ICR-8.1 murine V_(K) amino acid sequence was aligned withconsensus sequences of human V_(K) groups and it showed greatesthomology (79%) to human subgroup II. An initial humanized V_(K) was madeby taking an M13 clone containing the murine V_(K) DNA and mutating themurine framework DNA to a consensus sequence of human subgroup II (Kabatet al. supra). Each framework region was mutated by PCR using:

FR1 primer 33 (SEQ ID NO: 59)

GCTCTCCAGGAGTGACAGGCAGGG,

FR1 primer 36 (SEQ ID NO: 60)

TTGCGGCCGCAGGTGTCCAGTCCGACATTGTAATGACCCAGTC

TCCACTCTC,

FR1 primer 32 (SEQ ID NO: 61)

TCACTCCTGGAGAGCCAGCCTCCATCTCTTGCAGA,

FR3 primer 35 (SEQ ID NO: 62)

CCTCAGCCTCCACTCTGCTGATCTTGAGTGT,

FR3 primer 34 (SEQ ID NO: 63)

AGAGTGGAGGCTGAGGATGTGGGAGTTTATTACTGCTCTC, and

FR4 primer 37 (SEQ ID NO: 64)

TTGGATCCTAAGTACTTACGTTTTATTTCCACCTTGGTCCCCT GTCCG.

Mixtures for the PCR consisted of 0.2 μl supernatant of an M13 clonecontaining the ICR-8.1 murine V_(K) 25 pmol each oligonucleotide withother components as listed above in Section A.2. The samples weresubjected to 15 thermal cycles of 94° C., 30 seconds; 40° C., 30seconds; 72° C., 30 seconds. Product DNAs were of the expected size andwere joined in a second PCR to obtain full-length V_(K) usingoligonucleotides 36 and 37. The product DNA was cut with NotI and BamHIand cloned into M13 BM21. A fully humanized Vk with no spuriousmutations was identified by DNA sequencing. The sequences of this DNAand encoded product designated ICR-8.1 HuVK are shown in SEQ ID NOs: 65and 66.

5. Chimeric ICR-8.1 Antibodies

Chimeric antibodies consisting of murine V domains with human constantdomains were made for use as controls in subsequent assays whereinbinding affinities of CDR-grafted V regions were compared to parentalantibodies using a common anti-human Ig Fc antibody as a detectingreagent. NotI and BamHI sites were introduced into an M13 clonecontaining ICR-8.1 murine VH cDNA obtained in Section A.2 using in PCRoligonucleotides 27 (TTGCGGCCGCAGGTGTCCAGTCCGAGGTGCAACTGCAGCAGTCTGGAC,SEQ ID NO: 67) and 28 (TGGATCCAAGGACTCACCTGAGGAGACGGTGACTGAGGTTCC, SEQID NO: 68). Conditions for the PCR were as described in Section A.2except that there were 20 thermal cycles of 94° C., 30 seconds; 30° C.,30 seconds; 72° C., 45 seconds. The amplified DNA was cut with NotI andBamHI, cloned into M13 BM21 and a clone containing the desired sequenceidentified by DNA sequencing. This V_(H) differs from the murine V_(H)in that amino acids 2 and 7 are valine and serine respectively, insteadof the authentic residues Ile an Thr. This V_(H) is designated ICR-8.1MuVHVS.

Residue 28 in the murine V_(H) is a serine and is part of the structuralloop encompassing CDR1 and might be expected to play a role in antigenbinding. The corresponding residue in the human subgroup I consensus FRis Thr. In order to minimize the reintroduction of murine residues intothe humanized V_(H) the role of Ser28 in binding was assessed by makinga murine V_(H) region containing threonine at this position. Thr28 wasintroduced into MuVHVS DNA in a PCR essentially as described above inSection A.3 using in the first PCRs oligonucleotides 40(TTCTGGTTATACTTTCACTGACT, SEQ ID NO: 69) with the M13 universalsequencing primer and oligonucleotide 41 (AGTCAGTGAAAGTATAACCAGAA, SEQID NO: 70) with the M13 reverse sequencing primer. The two amplifiedDNAs were joined in a second PCR and the product subsequently cut withNotI and BamHI, cloned into M13 BM21 and a clone containing the Thr28mutation identified by DNA sequencing. This DNA is designated ICR-8.1MuVHVST.

A true chimeric V_(H) (designated MuVH) was constructed containing Ile2and Thr7. This was accomplished by PCR overlap/extension mutagenesisusing oligonucleotides 79 (CTCCGAGATCCAGCTGCAGCAGACTGGACC, SEQ ID NO:71) and 91 (CAGCTGGATCTCGGAGTGGACACCTGTGGAGAGAAAGGCAAAGTGG ATG, SEQ IDNO: 72). V_(K) for the chimeric kappa chain was constructed from an M13clone containing ICR-8.1 V_(K) cDNA obtained in Section A.3 above.Appropriate NotI and BamHI restriction sites were introduced by PCRusing oligonucleotides 43 (TTGCGGCCCGCAGGTGTCCAGTCCGACGCTGTGACCCAAAC,SEQ ID NO: 73) and 44 (TTGGATCCTAAGTACTTACGTTTTATTTCCAGCTTGGT, SEQ IDNO: 74) with conditions as described in Section A.2. Amplified DNA wascut with NotI and BamHI, cloned into M13 BM21 and a clone containing thecorrectly mutated DNA identified by DNA sequencing. This DNA wasdesignated ICR-8.1 MuVK and encodes the authentic N-terminal aminoacids.

6. Vectors for the Expression of Recombinant ICR-8.1 Antibodies

The heavy and light expression vectors were based on pSVgpt andpSVhygHuCK respectively Orlandi et al., Proc. Natl. Acad. Sci. USA, 86:3833-3837 (1989)! modified to include a NotI site in the intron betweenthe two exons encoding the signal peptide. Both vectors contain animmunoglobulin promoter and enhancer, signal sequence, appropriatesplice sites, the SV40 promoter and the gpt or hygromycin resistancegene for selection in mammalian cells and genes for replication andselection in E. coli. For expression of the humanized or chimeric heavychain the NotI-BamHl fragment containing the humanized V_(H) (HuVH,HuVHV or miHuVH) or murine VH (MuVHVS or MuVHVST) was cloned intoNotI-BamHI cut pSVgpt. For expression of MuVH, miHuVHI and miHuVHITheavy chains the V regions were cloned in as HindIII-BamHI fragmentsinto HindIII-BamHI cut pSVgpt. A human IgG₄ constant region Flanagan etal., Nature, 300:709-713 (1982)! was then added as a BamHI fragment. Forthe expression of the humanized or chimeric kappa chain the NotI-BamHIfragment containing the humanized V_(K) (HuVK) or murine V_(K) (MuVK)was cloned into NotI-BamHI cut pSVhygHuCK which contains DNA encodingthe human kappa chain constant region Hieter et al., Cell, 22:197-207(1980)!.

7. Expression of Recombinant ICR-8.1 Antibodies

The host for the expression of recombinant antibodies was either themouse myeloma NSO (ECACC 85110503) or the rat myeloma YB2/0 (ATCC CRL1662) and were grown in RPMI 1640 or DMEM plus 10% FBS. Cells in thelogarithmic phase of growth were harvested by centrifugation andresuspended in medium at 10⁶ cells per ml. 0.5 ml aliquots of cells weremixed with 10 μg of PvuI cut heavy chain expression vector and 20 μg ofPvuI cut light chain expression vector in ice for 5 minutes in anelectroporation cuvette. The cells were electroporated at 170 V, 960 μFusing a GenePulser apparatus (Biorad, Richmond, Calif.). After 20minutes in ice the cells were added to 20 ml growth medium and allowedto recover for 24-48 hours. At this time the cells were put into 50 mlgrowth medium containing 0.8 μg/ml mycophenolic acid and 200 μg/mlxanthine and 250 μl aliquots distributed into two 96-well plates. Thesewere incubated for 10-14 days at which time gpt⁺ colonies were visible.Supernatant from the wells was then assayed for the presence of humanIgG_(K) antibodies. Micro-titer plates (Immulon 4, Dynatech) were coatedwith goat anti-human IgG (Fc) or goat anti-human IgG (H+L) antibodies(Jackson Immunoresearch) and culture supernatant applied for 1 hour atambient temperature. After washing with PBST, captured human antibodywas detected with peroxidase-conjugated goat anti-human kappa (Sigma) orperoxidase-conjugated goat anti-human IgG (Fc) (Jackson Immunoresearch)antibodies. The substrate for peroxidase activity was o-phenylenediamineat 0.4 mg/ml in 50 mM citrate buffer pH 5.0 and 0.003% (v/v) H₂ O₂.Reactions were stopped by the addition of 50 μl 12.5% (v/v) sulphuricacid. The absorbance at 490 nm was then measured.

8. Purification of Recombinant ICR-8.1 Antibodies

Transfectants secreting recombinant ICR-8.1 antibody were expanded andgrown to saturation in RPMI 1640 plus 2% FBS or DMEM plus 11% FBS in 175cm² flasks. Culture medium was made 0.1 M Tris HCl pH 8.0 and wasstirred overnight with protein A agarose (Boehringer Manngeim) at about1 ml per liter medium. The protein A agarose beads were packed into asmall column, washed with 10 ml 35 mM Tris HCI pH 8.0, 150 mM NaCl, 0.1%(v/v) Tween 20 and then with 4 ml 50 mM citrate pH 5.0. Antibody waseluted with 1 ml amounts of 50 mM citrate pH 3.0, 0.02% (v/v) Tween 20.Samples were immediately neutralized with 1 M Tris HCl pH8.0 and theA₂₈₀ mm measured. Antibody containing fractions were pooled and dialyzedagainst PBS. Concentrations were determined spectrophotometrically.

9. Binding of Humanized ICR-8.1 Antibodies to ICAM-R

The binding of recombinant antibodies to ICAM-R was assessed by ELISA.Wells of a micro-titer plate (Immulon 4, Dynatech) were coated with 50ng per well baculovirus-produced soluble human ICAM-R in 50 or 100 μl 35mM sodium carbonate, 15 mM sodium bicarbonate pH 9.2 at 37° C. for 1hour or 16 hours at 4° C. Purified antibody or culture supernatantcontaining recombinant antibody was added and its binding detected asdescribed in section A.7 above.

Antibodies comprising the following combinations of humanized andchimerized chains showed equivalence in binding to ICAM-R: MuVHVS/MuVK,MuVHVST/MuVK, HuVH/MuVK, HuVHV/MuVK, HuVH/HuVK, miJuVH/MuVK andmiHuVH/HuVK. However, antibodies containing the humanized heavy chainwith the Ile2 mutation show an approximate 2-fold improvement in bindingover the aforementioned antibodies. In experiments in blocking thebinding of biotinylated murine ICR-8.1 to ICAM-R, the miHu/VHI/HuVK andmiHuVHI/MuVK antibodies compete about 2-fold less well than the murineantibody itself.

B. Humanization of ICR-1.1

1. RNA Isolation

ICR-1. 1 hybridoma cells were grown to subconfluency. One×10⁷ cells werelysed in 2.5 ml 1 M guanidine thiocyanate, 80 mM sodium acetate pH4.0,6.25 M sodium citrate, 40% (v/v) phenol, 16% (v/v) chloroform, 0.28%(v/v) 2-mercaptethanol and 0.125% (v/v) Sarkosyl. After isopropanolprecipitation the RNA was collected by centrifugation, dissolved inwater, ethanol precipitated, washed in 70% ethanol, dried and dissolvedin water. Yield of RNA measured spectrophotometrically was 0.16 mg.

2. Isolation of ICR-1.1 V Region cDNA

Heavy and light chain cDNA was made from random-primed ICR-1.1 RNA. Thereaction mixture was as in Section A.2 except that 10 μg RNA, 200 ngrandom primers pd(N)₆ ! and 44 u AMV reverse transcriptase were used andincubations were at 65° C. for 2 minutes, and after addition of theenzyme, at 42° C. for 1.5 hours.

V_(H) cDNA was amplified by PCR using a primer,

HG2A-1 (SEQ ID NO: 75)

GCTATCGGATCCGGARCCAGTTGTAYCTCCACACAC

based on amino acids 127-136 of the murine IgG2A constant region (Kabatet al., supra) and including a BamHI site for cloning purposes, togetherwith primers HFR1-1, -2, -3, -4, and -6 which are set out above inSection A.2 and

HFR1-5 (SEQ ID NO: 76)

CGATACGAATTCSAGGTSMARCTGCAGSAGTCT.

V_(K) cDNA was amplified using LKC-1 with either LFR1-1, LFR1-2, LFR1-3or LFR1-4. The sequences of these primers are listed in Section A.2. Theconditions for the PCR were essentially as in Section A.2 except thatthe samples were subjected to 25 thermal cycles of 95° C., 1 minute; 55°C., 2 minutes; 72° C., 3 minutes. V_(H) DNA of the expected size wasobtained with HFR1-1, -2, -3, -4 with HG2A-1, V_(K) DNA of the expectedsize was obtained using LKC-1 with LFR1-2, -3 and -4. Amplified DNAswere cut with BamHI and EcoRI, cloned into Bluescript SK+ and sequenced.Full length functional V region sequences were obtained. By comparisonwith other murine V regions, the ICR-1. 1 murine V_(H) and V_(K) geneswere members of murine heavy chain subgroup IIB and murine kappasubgroup VI (Kabat et al., supra). In order to determine the authenticN-terminal sequences of both heavy and kappa V regions, PCRs wereperformed with oligonucleotides based on known signal sequences of themurine heavy and kappa chain subgroups.

The murine ICR-1. 1 V_(H) sequence and its translation product are shownin SEQ ID NOs: 77 and 78, while the murine V_(K) sequence and itstranslation product are shown in SEQ ID NOs: 79 and 80.

3. Humanized ICR-1. 1 V_(H)

The murine ICR-1. 1 V_(H) amino acid sequence was aligned with consensussequences of human V_(H) subgroups where it shows closest homology (66%)to human subgroup I (Kabat et al., supra). Therefore, a consensus FRamino acid sequence of human heavy subgroup I was used as the recipientfor the ICR-1.1 CDRs. The template for the mutagenesis was uracilcontaining single-stranded DNA encoding a consensus sequence of humanheavy chain subgroup I containing irrelevant CDRs and cloned in M13.Mutagenesis reactions were as described above in Section A.3 usingmutagenic oligonucleotides for

CDR1 (SEQ ID NO: 81)

CCTGTCGCACCCAGTGCATCCAGTAAACAGTGAAGGTGTATCC;

CDR2 (SEQ ID NO: 82)

GTCCGCGGTAATGGTCACTCTGTCCTGGAACCTCTGATTGTACTC

AGTATAATCAGTGTTAGGATTAATGTATCCMATCCACTCGAGCCC;

and

CDR3 (SEQ ID NO: 83)

GGCCCCAGTAGTCCAAACCATAGGAGTTACCCCCCCATCTGG

CACAGTAATACACGG.

Amplified DNA was cut with NotI and BamHI, cloned into M13 BM21 and ahumanized VH clone identified by DNA sequencing. This clone wasdesignated M13 ICR-1.1 HuVHI and contains the murine framework aminoacid Ile48. Another variant that has the human amino acid methionine atposition 48 was made by the PCR overlap extension method as described inSection A.3 using in the first PCRs oligonucleotide 17(CTCGAGTGGATGGGATACATTAA, SEQ ID NO: 84) and the CDR2 oligonucleotidewith, respectively, the M13 universal and reverse sequencing primers.DNA after the second joining PCR was cut with NotI and BamHI, clonedinto M13 and a clone containing DNA encoding Met48 was identified by DNAsequencing. This DNA was designated ICR-1.1 HuVH and its DNA and encodedamino acid sequence are shown in SEQ ID NOs: 85 and 86.

An additional humanized V_(H) was made with a Ser73 to Lys73 mutation inframework region 3. This HuVHK was made by mutagenesis using PCRoverlap/extension of HuVH DNA and oligonucleotides 8(GTGGATCCAAGGACTCACCTGAGGAG, SEQ ID NO: 87) with 89(ACCGCGGACAAATCCACGAG, SEQ ID NO: 88) and 90 (CTCGTGGATTTGTCCGCGGT, SEQID NO: 89) with 92 (CACAGGTGTGTCCACTCCCAAGTCCAGC, SEQ ID NO: 90). Thetwo products were joined in a second PCR using oligonucleotides 8 and92. The product of this PCR was cut with HindIII and BamHI, cloned intoM13 and the desired clone identified by DNA sequencing.

4. Humanized ICR-1.1 V_(K)

The murine ICR-1.1 V_(K) amino acid sequence was compared with consensussequences of human V_(K) subgroups. It shows closest homology (62%) tohuman V_(K) subgroup I (Kabat et al., supra). Therefore, a consensus FRamino acid sequence of human kappa subgroup I was chosen as acceptoronto which the murine ICR-1.1 CDRs were grafted. The template for themutagenesis was uracil containing single-stranded DNA encoding aconsensus sequence of human kappa subgroup I (Kabat et al., supra)containing irrelevant CDRs and cloned in M13. Mutagenesis reactions wereas described in section using the following mutagenic primers:

CDR1 (SEQ ID NO: 91)

TTCTGTTGGTACCAGTAAATGTAACTTACACTTGAGCTGGCACTG

CAAGTGATGGTGAC;

CDR2 (SEQ ID NO: 92)

TTGATGGGACCCCAGAAGCCAGGTTGGATGTAAGATAGATCA

GGAGC;

CDR3 (SEQ ID NO: 93)

CCCCTGGCCGAACGTGAGTGGGATACTCTTCCACTGCTGACA

GTAGTAAGTTG.

One mutagenesis reaction included a fourth oligonucleotide(GTGAGAGTGTAGTCTGTCC, SEQ ID NO: 94) which would mutate Phe7l totyrosine. Amplified DNA was cut with NotI and BamHI, cloned into M13BM21 and humanized V_(K) DNAs identified by DNA sequencing. The clonesobtained were designated M13 ICR-1.1 HuVK and M13 ICR-1.1 HuVKY(containing Tyr71). The DNA and deduced amino acid sequences of HuVK areshown in SEQ ID NOs: 95 and 96.

An additional humanized Vk was made containing the murine N-terminalamino acids Glu1 Val3 Leu4. Mutagenesis of HuVK to HuVKQVL wasaccomplished by PCR using oligonucleotide 110(CACAGGTGTCCACTCCCAAATCGTGCTGACCCAGTCTCCATCCTCCC, SEQ ID NO: 97) and 68(TTAAAGATCTAAGTACTTACGTTTGATCTC, SEQ ID NO: 98). The DNA product wasjoined to DNA amplified with oligonucleotides 12 and 82 containing theimmunoglobulin promoter and signal sequence. The full-length DNA was cutwith HindIII and BamHI, cloned into M13 and the desired clone identifiedby DNA sequencing.

5. Chimeric ICR-1.1 Antibodies

Chimeric antibodies consisting of murine V domains with human constantdomains were constructed to act as appropriate controls. Forconstruction of the murine VH as a HindII-BamHI fragment containing theimmunoglobulin promoter and signal sequence, an M13 clone with themurine VH was subject to PCR with oligonucleotides 92(CACAGGTGTCCACTCCCAAGTCCAGC, SEQ ID NO: 99) and 93(TTGGATCCAAGGACTCACCTGAGGAGACGGTGACTGAGGT, SEQ ID NO: 100). The PCRproduct was joined to that resulting from the amplification of theimmunoglobulin promoter and signal sequence using oligonucleotides 12and 82. The product DNA was cut with HindIII and BamHI and the correctclone identified by DNA sequencing.

For construction of the chimerized V_(K), an M13 clone containing MuVKwas subject to a PCR using oligonucleotides 87(TTGGATCCTAAGTACTTACGTTTCAGCTCCAGCTTGGTCCCAG 3', SEQ ID NO: 101) and 88(CAGGTGTCCACTCCCAAATTGTTCTCACCCAGTCTCCAGCACTCATG, SEQ ID NO: 102). Theproduct was also joined to DNA amplified with 12 and 82. The resultingDNA was cut with HindIII and BamHI, cloned into M13 and the desiredclone identified by DNA sequencing.

6. Vectors for the Expression of Recombinant ICR-1.1 Antibodies

The CDR-grafted humanized heavy and kappa chain V region DNAs were cutwith NotI and BamHI and cloned into the pSVgpt and pSVhyg HuCKexpression vectors as described above in Section A.7. Where therecombinant V regions were constructed with the immunoglobulin promoterand signal sequence these DNAs were cut with HindIII and BamHI andcloned into HindIII and BamHI cut pSVgpt and pSVhygHuCK as appropriate.A human IgG4 constant region was added to the heavy chain vectors asdescribed above in section A.6.

7. Expression of Humanized ICR-1.1 Antibodies

Transfection of vectors into YB2/0 and NSO cells and the isolation ofantibody-secreting clones was as described in above section A.7.

8. Purification of Humanized ICR-1.1 Antibodies

Recombinant ICR-1.1 antibodies were purified as described above insection A.8.

9. Binding of Recombinant ICR-1.1 Antibodies to ICAM-R

Binding of humanized or chimeric ICR-1.1 antibodies to solublebaculovirus produced human ICAM-R (Example 9) was determined by ELISA asdescribed in Section A.9 above. Analysis of the binding of the chimeric,humanized and hybrid antibodies indicates that compared with theirmurine progenitors the HuVH and HuVK versions show 2-3 fold and 2-folddeficits respectively. The HuVH/HuVK antibody is thus 5-10 fold lesseffective in binding to ICAM-R than the chimeric (MuVH/MuVK) antibody.Antibodies containing HuVHI, HuVKY or HuVKQVL show no increase inaffinity whereas those containing HuVHK show a possible 1.5-foldimprovement.

Because both HuVH and HuVK showed deficiencies compared to the murineheavy and light chains, in an attempt to improve avidity of theHuVH/HuVK antibody, four heavy chain variants and one light chainvariant were made by similar methods to those described above. Thefollowing amino acid changes were made: Arg66 Val67 to Lys66 Ala67 togenerate a heavy chain designated HuVHKA, Thr73 to Lys73 to generate aheavy chain designated HuVHK, Thr75 to Ser75 to generate a heavy chaindesignated HuVHS, Ala40 to Arg40 to generate a heavy chain designatedHuVHR, and Leu46 to Pro46 to generate a light chain designated HuVKP.

In ELISAs measuring binding to baculovirus produced ICAM-R (Example 9)the mutations at positions 40 in HuVHR, 66 in HuVHKA, 67 in HuVHKA and75 in HuVHS did not affect binding, while the change at position 73 inHuVHK improved avidity 1.5 to 2-fold. The HuVKP light chain with themutation at position 46 also displayed an increase in avidity.HuVHK/HuVKP antibody is within 50% of the chimeric antibody (MuVH/MuVK)in binding to baculovirus produced ICAM-R (Example 9) or ICAM-R/Igfusion protein (Example 25) as measured by ELISA. However, incompetition experiments, 5 to 10-fold more of the HuVHK/HuVKP antibodyis required to compete equally against the murine or chimerizedantibodies. Therefore, the mutations at position 73 in the heavy chainand position 46 in the light chain do increase the binding capacity ofHuVHK/HuVKP over HuVH/HuVK antibody.

EXAMPLE 14

FACS-based competition assays utilizing human peripheral bloodleukocytes or SKW3 cells (both ICAM-R expressing cells) indicate thatmonoclonal antibodies ICR-4.2 and ICR-1.1 are immunologically reactivewith distinct epitopes of ICAM-R.

In the assays, human peripheral blood leukocytes (PBL) obtained byFicoll Hypaque centrifugation of normal peripheral blood were washedtwice in ice cold FACS buffer (PBS containing 0.1% sodium azide and 1%bovine serum albumin) and 2×10⁵ cells were incubated in triplicatepolypropylene tubes with 5 μg of each of the following antibodiesICR-1.1, ICR-4.2, and control isotype IgG (Sigma). All tubes containingthe first stage antibodies were then incubated for 30 minutes at 4° C.and washed twice in cold FACS buffer. To each triplicate tube, 5 μg ofeach of the following second stage antibodies were added:biotinylated-ICR-1.1, biotinylated-ICR-4.2, biotinylated-anti-rat CD4(negative control). All second stage antibodies were biotinylatedaccording to standard procedures as described in Example 12 and alltubes were then incubated for an additional 30 minutes at 4° C. beforewashing twice in FACS buffer. Five ul of a 1:10 dilution ofStrepavidin-phycoerythrin (Southern Biotechnology, Birmingham, Ala.) wasthen added to each tube containing 50 ul FACS buffer and all tubes wereincubated for 30 minutes at 4° C. Finally, all tubes were washed twicein FACS buffer and analyzed by flow cytometry (FACScan,Becton-Dickinson).

While monoclonal antibody ICR-4.2 blocked binding ofbiotinylated-ICR-4.2 to ICAM-R on PBL, it did not block binding ofmonoclonal antibody ICR-1.1. Similarly, monoclonal antibody ICR-1.1 didblock binding of biotinylated-ICR-1. 1 but did not block binding ofmonoclonal antibody ICR-4.2. These results indicate that the twoantibodies recognize distinct epitopes on ICAM-R. Equivalent resultswere obtained when using the human cell line SKW3 as follows. SKW3 cellswere labelled with either 1 μg of antibody ICR-1.1 or ICR-4.2, washed inFACS buffer and incubated with 1 μg biotinylated-ICR-1.1 or biotinylatedICR-4.2. All tubes were then washed in FACS buffer, incubated withStrepavidin-phycoerythrin for an additional 30 minutes at 4° C. andanalyzed by FACScan.

In the assays, if an unlabeled antibody (the "blocking" antibody)prevented the labelled antibody from binding to ICAM-R, it indicatesthat the unlabeled antibody "competes" with the labelled antibody forbinding to ICAM-R and that the two antibodies recognize the same,sequential or sterically overlapping epitopes on ICAM-R. A variation ofthe competition assay in which unlabeled antibody is used to "competeaway" binding of a labelled antibody may also be utilized to determineif two antibodies recognize the same, sequential or stericallyoverlapping epitopes.

The specific ICAM-R epitopes recognized by the various monoclonalantibodies of the invention can be mapped by four different methods.

A. Epitope Mapping Using The Multipin Peptide Synthesis System

The first method for mapping linear epitopes recognized by the ICAM-Rspecific antibodies of the invention utilized the Multipin PeptideSynthesis System (Chiron Mimotopes Pty. Ltd., Victoria, Australia) whichplaces ten amino acid peptides representing overlapping segments of theprotein of interest on the surface of a series of plastic pins. Amodified ELISA test is performed to determine binding of a monoclonalantibody to each peptide.

The ELISA to determine binding of the monoclonal antibodies to ICAM-Rpeptides was run as follows. The pins were placed in five 96-well platescontaining 200 μl per well blocking buffer (2% weight/volume BSA, 0.1%volume/volume Tween 20, 0.01M PBS, pH 7.2) and incubated for one hour at20° C. with agitation. The pins were transferred to plates with 175 μlper well of undiluted anti-ICAM-R monoclonal antibody supernatant andincubated overnight at 4° C. with agitation. The pins were then washedfour times with 0.01M PBS, pH 7.2 (10 minutes/wash at 20° C. withagitation) and placed in plates containing 175 μl per well HRP-Goatanti-mouse IgG (H+L) (Kirkegaard and Perry Laboratory Inc.,Gaithersburg, Md.) diluted to an appropriate concentration in conjugatediluent (1% volume/volume sheep serum, 0.01% volume/volume Tween 20,0.1% weight/volume sodium caseinate and 0.01M PBS). The plates wereagitated for one hour at 20° C., and washed four times with 0.01M PBS.The pins were transferred to plates containing ABTS substrate solution0.5 mg/ml ABTS, 0.01% weight/volume H₂ O₂ in substrate buffer (17.9 g/LNa₂ HPO₄ H₂ O, 16.8 g/L citric acid monohydrate, pH 4.0)! for 45 minutesat 20° C. with agitation and then the plates were read at 410/495 nm.

Relative reactivity with individual pins was determined afternormalizing results for differences in immunoglobulin concentrations inanti-ICAM-R and control hybridoma supernatants and reactivities ofpositive controls between assays. Mouse IgG levels for each supernatanthad been determined by antibody capture ELISA as follows. Immulon 4plates were coated and washed as described in Example 10C. Fifty μl/wellof culture supernatant diluted in PBST or known concentrations indoubling dilutions in PBST of mouse IgG₁, and IgG_(2a) (MOPC-21, andUPC-10) (Sigma)! was added to the plate. After incubating for 1 hour atroom temperature and washing 3 times with PBST, horseradish peroxidaseconjugated goat anti-mouse IgG(fc) (Jackson ImmunoResearch, West Grove,Pa.) was diluted 1:2000 for mouse IgG₁ and 1:1000 for IgG_(2a), andadded 50 μl/well. After the plate was incubated for 1 hour at roomtemperature and washed 4 times in PBST, the remainder of the assay wasconducted as described in Example 10C. Antibody concentrations ofculture supernatant were determined by fitting measured opticaldensities to the standard curve of the isotype matched control.

Strong reactivity of monoclonal antibody ICR-1.1 was noted with twooverlapping peptides spanning amino acids 13-23, as illustrated below:

SEQ ID NO: 103

VLSAGGSLFV

SEQ ID NO: 104

LSAGGSLFVN

Regions reactive with anti-ICAM-R antibodies can also be defined and/orverified using the following methodologies.

B. Epitope Mapping Using A Library of Bacterial Clones

Epitope mapping with the anti-ICAM-R antibodies was also performed usingthe Novatope Library Construction and Screening System (Novagen,Madison, Wis.). Using this method, a library of bacterial clones isgenerated wherein each clone expresses a polypeptide including a smallpeptide derived from the protein being examined. The library is thenscreened by standard colony lift methods using monoclonal antibodies asprobes.

Double-stranded DNA encoding the external domain of ICAM-R (amino acids1 to 487) from pVZ147 (See Example 4) was cut with different amounts ofDNAseI in the presence of 10 mM manganese for 10 minutes at 21 ° C. Thereaction was stopped with EDTA and 1/10 of the reaction waselectrophoresed on a 2% agarose gel with ethidium bromide andappropriate markers. Those reactions containing fragments in the 50-150bp range were pooled and electrophoresed on another 2% gel. The area ofthe gel between 50-150 bp was excised, the fragments contained thereinwere electroeluted into dialysis tubing (SP Brand Spectra/Por 2, MWCO12-14,000), and then phenol/chloroform extracted and ethanolprecipitated.

One μg DNA was blunted according to the manufacturer's protocol, usingT4 DNA polymerase and all four dNTPs. The reaction was stopped byheating to 75° C. for 10 minutes, then a single 3' dA residue was addedby using Tth DNA polymerase (Novagen). The reaction was stopped byheating to 70° C. for 15 minutes and extracted with chloroform. Whenstarting with 1 μg of DNA, the final concentration was 11.8 ng/μl in 85μl. The dA tailed fragments are ligated into the pTOPE T-vector(Novagen) which is designed for the expression of inserts as stablefusion proteins driven by T7 RNA polymerase (the structural gene forwhich is carried on a replicon in the host cell). Using 6 ng of 100 bpDNA (0.2 pmol), the ligation reaction was run at 16° C. for 5 hours.NovaBlue(DE3) (Novagen) cells were transformed with 1 μl (1/10) of thereaction mix, and spread on LB agar (carbenicillin/tetracycline) platesto obtain an initial count of transformants. The remainder of theligation reaction was put at 16° C. for an additional 16 hours. Based onthe initial plating, 2 μl of the ligation reaction was used to transform40 μl of competent NovaBlue(DE3) cells, then 8 plates were spread at adensity of approximately 1250 colones/plate for screening with antibody.

Colonies were screened using standard colony lift methods ontonitrocellulose membranes, lysed in a chloroform vapor chamber anddenatured. Using anti-ICAM-R monoclonal antibody ICR-1.1 at a 1:10dilution in TBST (Tris-buffered saline/Tween) as a primary antibody, theassay was developed using an alkaline phosphatase-coupled secondaryreagent. The substrate mix was incubated for 30 minutes. One isolatedcolony gave a strong positive reaction. Three others areas (not isolatedcolonies) gave weak positive reactions. Streaks were made from a stab ofthe isolated colony or colony areas for re-screening. Upon re-probingwith ICR-1.1, the streak from the isolated colony had positive reactiveareas after a 20 minute incubation with substrate. The other threecolony area samples were negative. A stab from the ICAM-R reactive areawas re-streaked, incubated overnight at 37° C. and re-probed incubatingwith substrate for 10 minutes. Many ICR-1.1 reactive colonies resulted.Plasmid DNA recovered from these colonies can be sequenced and the aminoacid sequence corresponding to the ICR-1.1 reactive epitope can bedetermined.

C. Epitope Mapping by Domain Substitution-Construction of ChimericICAM-R Molecules and Deletion Mutants

Conformational epitopes of ICAM-R recognized by the monoclonalantibodies of the invention may be mapped by domain substitutionexperiments. In these experiments, chimeric variants of ICAM-R aregenerated in which selected immunoglobulin-like domains of ICAM-R arefused to portions of ICAM-1 and assayed for binding to the monoclonalantibodies of the invention by FACS.

FIG. 7 is a diagram of the chimeric proteins whose construction isoutlined below. Protein number 1 contains the amino-terminalimmunoglobulin-like domain of ICAM-R (residues 1 to 93) fused to ICAM-1(residue 117 to 532). Protein number 2 contains the first two aminoterminal immunoglobulin-like domains of ICAM-R (residues 1 to 190) fusedto ICAM-1 (residues 216 to 532). Protein number 3 contains the firstthree immunoglobulin-like domains of ICAM-R (residues 1 to 291) fused toICAM-1 (residues 317 to 532).

1. Chimeric Protein 1

Protein number 1 was made by engineering a unique Nhe I site into thecoding sequences of ICAM-R and ICAM-1 at the junction ofimmunoglobulin-like domains 1 and 2 of each. The DNA sequence of ICAM-Rwas subcloned into the M13 BM21 vector (Boehringer) as described inExample 9 resulting in a molecule called M13 BM21ICAM-R. The entirecoding sequence of ICAM-1 Simmons et al., Nature, 331:624-627 (1988)!was subcloned into the plasmid pBSSK(+) (Stratagene). The resultingplasmid, pBSSK(+)ICAM-1 was cut with SalI and KpnI to release the ICAM-1coding sequence along with a short segment of the multiple cloning sitesand ligated to M13 BM21 cut with restriction enzymes SalI and KpnIresulting in a molecule called M13 BM21ICAM-1. M13 phage isolates wereverified by DNA sequence analysis.

Mutagenizing oligonucleotides ICAM1.D1. Nhe 1 (corresponding tonucleotides 426 to 393 of ICAM-1) and ICAMR.D1Nhe 1 (corresponding tonucleotides 367 to 393 of ICAM-R) having the following sequences weresynthesized by routine laboratory methods:

ICAM1.D1.NheI (SEQ ID NO: 105)

AGAGGGGAGGGGTGCTAGCTCCACCCGTTCTGG

ICAMR.D1.NheI (SEQ ID NO: 106)

GAGCGTGTGGAGCTAGCACCCCTGCCT

Nucleotides 16 and 19 of ICAM1.D 1.NheI and nucleotide 15 of ICAMR.D1.NheI form mismatch base pairs when the oligos are annealed to theirrespective complementary DNA sequences. Both oligonucleotides introducea recognition site for endonuclease Nhe I. Site-directed mutagenesiswith the oligonucleotides was employed to introduce the sequences ofthese oligos into the respective ICAM-1 and ICAM-R target DNA sequencesM13 BM21ICAM-1 and M13 BM21ICAM-R. Several phage isolates from eachmutagenesis reaction were sequenced to verify that the correct DNAsequence was present. These isolates were designated M13 BM21ICAM-R.NheIand M13 BM21ICAM-1.NheI.

The coding region for the ICAM-R signal peptide and immunoglobulin-likedomain 1 was isolated from M13 BM21ICAM-R.NheI by the following method.Ten μg of purified single strand M13 BM21ICAM-R.NheI phage DNA wasannealed to 50 ng Lac Z universal -20 primer (SEQ ID NO: 28 in 1× KlenowDNA polymerase buffer (10 mM Tris-Cl pH 7.5, 5 mM MgCl₂, 7.5 mMdithiothreitol) by incubating the mix at 65° C. for 5 minutes and then25° C. for 5 minutes. The following mixture was then added to theannealing reaction: 33 μM final concentration dATP, dGTP, dCTP, dTTP; 4units of Klenow DNA polymerase (Boehringer), and 1× Klenow buffer. Theprimer extension reaction was allowed to incubate at 37° C. for 45minutes prior to being stopped by a single phenol/chloroform (1:1)extraction and ethanol precipitation. The dried pellet was resuspendedin 1× EcoRI buffer and 20 units each of EcoRI and NheI endonucleaseswere added prior to a 60 minute incubation at 37° C. A 412 bp fragmentcontaining the coding sequence for ICAM-R signal peptide andimmunoglobulin-like domain 1 was agarose gel purified.

The DNA sequence of ICAM-1 containing the coding region forimmunoglobulin-like domains 2 through 5, the transmembrane andcytoplasmic domains was isolated by restriction enzyme digest. Ten μg ofprimer extended M13.BM21ICAM-1.NheI were cut with NheI and NotI. Thisresulted in the release of a DNA fragment of 1476 bp which was agarosegel purified.

Five μg of the mammalian expression plasmid pcDNAI/Amp (Invitrogen) wasdigested with EcoRI and NotI and purified by spin column chromatography.A 20 μl ligation mix was assembled containing the following components:50 ng linear pCDNA1Amp with EcoRI and NotI termini, 100 ng of the 412 bpICAM-R fragment, 100 ng of the 1476 bp ICAM-1 fragment, 1× ligase bufferand 1 unit of T4 DNA ligase (Boehringer). The reaction was incubated at25° C. for 16 hours and used to transform competent XL-1 cells (Biorad).Transformants were selected on LB plates supplemented with carbenicillinat a final concentration of 100 μg/ml. Transformants were analyzed usinga standard mini DNA prep procedure and digestion with diagnosticendonucleases. Isolates designated pCDNA1Amp.RD1.ID2-5 were chosen forexpression studies.

A chimeric gene encoding protein number 1 was also generated by analternative method as follows. An approximately 375 bp EcoRI-NheIfragment of ICAM-R containing domain 1 and an approximately 1500 bpNheI-NotI fragment of ICAM-1 containing the extracellular domains 2-5,the transmembrane domain and the cytoplasmic tail were gel purifiedafter restriction enzyme digestion of the double stranded RF(replicative form) DNA from the M13BM21ICAM-R and M13 BM21ICAM-1 clonesand agarose gel electrophoresis of the corresponding double strandedplasmid DNAs. The resulting two DNA fragments were cloned by a three wayligation into an EcoRI and NotI digested and calf intestinalphosphatase-treated expression vector pcDNAI/Amp (Invitrogen). E. coliXL1 blue (Stratagene) strain was transformed with the ligation mixtureand the transformants were selected on carbenicillin containing plate.Clones with the desired inserts were identified by restriction enzymedigestion of the plasmid DNA minipreps.

2. Chimeric Proteins 2 and 3

To construct coding sequences for proteins 2 and 3, engineered versionsof M13 BM21ICAM-1 and M13 BM21ICAM-R in which a unique NheI site wascreated between immunoglobulin-like domains 2 and 3 or a unique AflIIsite was created between immunoglobulin-like domains 3 and 4 weregenerated by methods similar to those described in the foregoingparagraphs. Four oligonucleotides (ICAM-1.D2.NheI corresponding tonucleotides 686 to 713 of ICAM-1, ICAM-R.D2.NheI corresponding tonucleotides 655 to 690 of ICAM-R, ICAM-1.D3.AflII corresponding tonucleotides 987 to 1026 of ICAM-1, and ICAM-R.D3.AflII corresponding tonucleotides 962 to 993 of ICAM-R) with the sequences set out below weresynthesized for this purpose.

ICAM-1.D2.NheI (SEQ ID NO: 107)

GGGGGAGTCGCTAGCAGGACAAAGGTC

ICAM-R.D2.NheI (SEQ ID NO: 108)

CGAACCTTTGTCCTGCTAGCGACCCCCCCGCGCCTC

ICAM-1.D3.AflII (SEQ ID NO: 109)

TGAGACCTCTGGCTTCCTTAAGATCACGTTGGGCGCCGG

ICAM-R.D3.AflII (SEQ ID NO: 110)

GACCCATTGTGAACTTAAGCGAGCCCACC

Nucleotide 13 of ICAM1.D2NheI; nucleotides 17, 18 and 20 ofICAMR.D2.NheI; nucleotides 17, 18, 20 and 22 of ICAM-1.D3. AflII; andnucleotides 15 and 17 of ICAM-R.D3.AflII form mismatch base pairs whenthe oligonucleotides are annealed to their respective complementary DNAsequences. The appropriate coding sequences of ICAM-R and ICAM-1(sequences encoding the first two amino terminal immunoglobulin-likedomains of ICAM-R fused to sequences encoding ICAM-1 residues 118 to 532for protein 2 and sequences encoding the first three immunoglobulin-likedomains of ICAM-R fused to sequences encoding ICAM-1 residues 317 to 532for protein 3) were then subcloned into expression plasmid pCDNA1Amp(Invitrogen) to generate isolates pCDNA1Amp.RD1-2.1D3-5 andpCDNAAmp.RD1-3.1D4-5 respectively encoding ICAM-R variant proteins 2 and3.

Gene fusions encoding protein numbers 2 and 3 were also constructed byalternative methods as follows.

For the generation of protein 2 encoding sequences, an NheI wasintroduced by oligonucleotide directed in vitro mutagenesis in betweendomains 2 and 3 in both ICAM-R and ICAM-1. An approximately 700 bpEcoRI-NheI fragment of ICAM-R containing the domains 1 and 2, and anapproximately 1100 bp NheI-NotI fragment of ICAM-I containing thedomains 3-5, the transmembrane domain and the cytoplasmic tail weresubcloned by a three-way ligation into the EcoRI and NotI digested andcalf intestinal phosphatase-treated pcDNAI/Amp plasmid DNA. For thegeneration of protein 3 encoding sequences an approximately 1000 bpNotI-AflII fragment of ICAM-R containing domains 1 through 3, and anapproximately 850 bp AflII-NotI fragment of ICAM-1 containing domains4-5, the transmembrane domain and the cytoplasmic tail were purified byrestriction enzyme digestion of the plasmid DNAs and agarose gelelectrophoresis. These two fragments were cloned by a three way ligationinto the NotI digested and phosphatase treated pcDNAI/Amp plasmid DNA.Clones containing the insert with the desired orientation wereidentified by restriction enzyme digestion of plasmid DNA minipreparations.

3. Domain Deletion Proteins

ICAM-R domain deletion variants were generated by similaroligonucleotide directed mutagenesis protocols as described above forchimeric protein numbers 1, 2 and 3. A domain 1 deletion variant whichlacks amino acids 2-90 of ICAM-R (SEQ ID NO: 1), a domain 1 and 2deletion variant which lacks amino acids 2-203, and a domain 3 deletionvariant lacking amino acids 188-285 were constructed.

Control plasmids containing the full length ICAM-R or ICAM-1 cDNAsequences were generated by ligating gel-purified cDNA fragments toplasmid pCDNA1Amp. The two plasmids pCDNA1AmpICAM-1 and pCDNA1AmpICAM-Rexpress the full length ICAM-1 and ICAM-R proteins, respectively, sothat monoclonal antibody binding to native protein in equivalentcellular contexts can be assessed.

COS cells were transfected with the plasmid DNA encoding the ICAM-Rchimeric or deletion mutant proteins or with the plasmid DNApCDNA1AmpICAM-1, pCDNA1AmpICAM-R or pCDNA1Amp by the DEAE-dextranmethod. Typically, the COS cells were seeded at a density of about 7.0×10⁵ cells on a 10 cm diameter plate and grown overnight in Dulbecco'smodified Eagles medium (DMEM) containing 10% fetal bovine serum (FBS).The next day the cell monolayer was rinsed with DMEM and exposed to 10ml of transfection mixture containing 10 ug of the desired plasmid DNA,0.1M chloroquine and 5.0 mg DEAE-dextran in DMEM for 2.5 hours at 37° C.After the incubation, the transfection mixture was aspirated and themonolayer was treated with 10% DMSO in PBS for one minute. The cellswere washed once with DMEM and incubated with DMEM containing 10% FBS.The next day the medium was replaced with fresh medium and theincubation was continued for two more days.

Expression of all the chimeric and deletion ICAM-R proteins wasobtained. The domain 1 and domain 3 deletion variants expressed at alevel of 50-60% compared to the wild type ICAM-R protein.

D. Epitope Mapping by Domain Substitution--Monoclonal Antibody BindingAssay

For the anti-ICAM-R monoclonal antibody binding assay, COS cellstransfected with constructs encoding the ICAM-R chimeric proteins orcontrol constructs were removed from the plates by EDTA treatment andaliquoted at 2.5×10⁵ cells per well in a 96-well round bottom plate.Cells were washed 3 times with ice cold washing buffer (PBS containing1% BSA and 0.05% sodium azide). Anti-ICAM-R monoclonal antibody wasapplied at 5.0 ug/ml in 50 ul final volume and incubated on ice for 30minutes. Cells were then washed three times with cold washing buffer andincubated with the FITC labeled secondary antibody (sheep anti-mouse IgGF(ab')₂) at a 1:100 dilution on ice for 30 minutes in dark in 50 ulfinal volume. After the incubation, cells were washed again for threetimes in the ice cold washing buffer and resuspended in 200 ul of 1%paraformaldehyde. The samples were analyzed on a Becton-DickinsonFACScan instrument. Results of the assay are given below in Table 5 aspercent positive COS cell transfectants, wherein MOPC 21 (IgG1) and UPC10 (IgG2a) are isotype matched controls, 18E3D is an ICAM-1 specificmonoclonal antibody and ICR-1.1 to ICR-9.2 are ICAM-R specificmonoclonal antibodies. The reactivities of monoclonal antibodies ICR-1.1through ICR-9.2 were assayed in a different experiment than monoclonalantibodies ICR-12. 1 through ICR-17.

                  TABLE 5                                                         ______________________________________                                        Molecule                                                                      Antibody                                                                              Protein 1                                                                              Protein 2                                                                              Protein 3                                                                            ICAM-R ICAM-1                                ______________________________________                                        MOPC 21 1.16     1.90     1.86   1.41   1.45                                  UPC 10  2.00     1.41     1.69   1.67   1.04                                  18E3D   1.24     1.23     1.14   1.60   39.99                                 ICR-1.1 60.27    68.32    52.71  54.33  2.43                                  ICR-2.1 50.77    60.06    43.97  49.50  1.94                                  ICR-3.1 56.73    63.09    47.78  50.13  1.90                                  ICR-4.2 1.80     55.38    42.05  44.40  1.47                                  ICR-5.1 58.30    62.38    48.43  48.42  1.85                                  ICR-6.2 2.36     52.55    42.48  41.28  1.19                                  ICR-7.1 47.54    41.76    37.78  38.33  1.43                                  ICR-8.1 57.34    64.25    44.93  48.85  1.08                                  ICR-9.2 2.12     66.84    46.64  50.69  2.39                                  ICR-12.1                                                                              70.73    71.73    55.14  58.92  ND                                    ICR-13.1                                                                              72.22    71.43    58.66  56.92  ND                                    ICR-14.1                                                                              72.40    70.45    54.51  56.60  ND                                    ICR-15.1                                                                              72.64    73.91    58.83  55.69  ND                                    ICR-16.1                                                                              72.59    74.09    55.01  59.06  ND                                    ICR-17.1                                                                              72.00    74.87    57.81  54.10  ND                                    ______________________________________                                    

The results presented above show that the antibodies ICR-1.1, 2.1, 3.1,5.1, 7.1, 8.1, 12.1, 13.1, 14.1, 15.1, 16.1 and 17.1 recognize thehybrid molecule in which only the ICAM-1 domain 1 has been replaced withthe ICAM-R domain 1. The antibodies ICR-4.2, 6.2 and 9.2 recognize themolecule in which a minimum of 2 domains (domain 1 and 2) of ICAM-1 wasreplaced with the corresponding domains of ICAM-R. Based on theseresults the antibodies have been categorized as either domain 1 ordomain 2 specific.

The ICAM-R chimeric and deletion mutant protein constructs can also beused to transfect rat L cells by a calcium phosphate co-precipitateprotocol using 10 μg of 2× CsCl-banded plasmid DNA. In this protocol,forty-eight hours post-transfection the cells are released from thedishes by mild trypsinization. The cells are divided and incubated onice with anti-ICAM-R monoclonal antibodies or a control isotype matchedmonoclonal antibody at a concentration of 10 μg/ml or no monoclonalantibody for 1 hour. The cells are then processed for FACS analysis aspreviously described in Example 12C.

E. Epitope Mapping by Amino Acid Substitution

Differential reactivity of an anti-ICAM-R antibody of the invention withthe ICAM-R variant proteins as described above thus is indicative ofreactivity with a specific domain of ICAM-R. Once particular domains areidentified that reacted with specific anti-ICAM-R monoclonal antibodies,individual residues within those domains are changed by oligo-directedsite specific mutagenesis to determine their relative effects onmonoclonal antibody binding. Based on computer algorithms that predictprotein hydropathy and secondary structure (Kyte et al., supra),particular residues that have the potential for antibody interactionsare targeted for mutagenesis.

Mutagenesis of ICAM-R was carried out according to the procedure ofKunkel et al., supra. E. coli strain Cj236 (dut ung) was transformedwith the plasmid pcDNA1/AmpICAM-R (see Section C above) byelectroporation. The transformants were selected on carbenicillincontaining plate. One of the transformants was infected with the helperphage M13K07 and grown overnight. Uracil-containing single stranded DNAwas prepared from the culture supernatant and used for mutagenesis.Mutagenic oligonucleotides were hybridized to the uracil containingsingle stranded DNA of pcDNA1/Amp-ICAM-R. Using the mutagenicoligonucleotides as primers, DNA synthesis and ligation reactions werecarried out using T7 DNA polymerase and T4 DNA ligase, respectively. Analiquote of the synthesis reaction was used to transform E. coli XL1blue (Stratagene) strain and transformants were selected oncarbenicillin containing plates. Growth of the uracil containing plasmidDNA in this strain markedly reduces the propagation of the uracilcontaining DNA (wild type) strand. Mutants were selected by plasmid DNAminipreps and diagnostic restriction enzyme digestion. Sequences werefurther verified by DNA sequence analysis. The mutations made were:F21V/AS, E32K/AS, K33I/AL, E37T/AS, T38/A, L40/A, K42E/AS, E43/A,L44V/AL, W51A/AS, R64/Q, S68/A, Y70/A, N72/Q, Q75I/AS, N81/Q. Mutation"F21V/AS" indicates, for example, that the phenylalanine at position 21of ICAM-R (SEQ ID NO: 1) and the valine at position 22 were respectivelychanged to an alanine and a serine, while mutation "T38/A" indicatesthat the threonine at position 38 of ICAM-R (SEQ ID NO: 1) was changedto an alanine. Effects of each mutation on anti-ICAM-R monoclonalantibody binding were tested according to the procedure described inSection C above. Table 6 below summarizes the results obtained, whereina mutation with a "critical" effect was defined as 0-20% binding of anantibody in comparison to binding to wild type ICAM-R, an "important"effect was defined as about 50% binding in comparison binding to wildtype ICAM-R, and a minor effect was defined as about 75% binding incomparison to binding to wild type ICAM-R. Mutations that did not effectbinding of an antibody are not listed in Table

                                      TABLE 6                                     __________________________________________________________________________    Effect                                                                        of Mutating                                                                   Amino Acid                                                                    Position(s)                                                                         Monoclonal Antibody                                                     on Binding                                                                          ICR1.1                                                                            ICR2.1                                                                            ICR3.1                                                                            ICR4.2                                                                            ICR5.1                                                                            ICR6.2                                                                            ICR7.1                                                                            ICR8.1                                                                            ICR9.2                                  __________________________________________________________________________    Critical                                                                            F21V                                                                              F21V                                                                              F21V                                                                              F21V                                                                              F21V                                                                              F21V                                                                              F21V                                                                              F21V                                                                              F21V                                    Critical                                                                            E32K                                                                              E32K                                                                              E32K                                                                              --  --  --  --  E32K                                                                              --                                      Critical                                                                            --  K33I                                                                              --  --  --  --  --  --  --                                      Critical                                                                            E37T                                                                              --  --  --  E37T                                                                              --  E37T                                                                              --  --                                      Critical                                                                            --  --  --  --  --  --  W51A                                                                              --  --                                      Critical                                                                            --  --  --  --  --  --  Y70 --  --                                      Critical                                                                            --  --  --  --  --  --  Q75I                                                                              --  --                                      Important                                                                           --  --  --  --  --  --  E32K                                                                              --  --                                      Important                                                                           K33I                                                                              --  --  --  --  --  --  --  --                                      Important                                                                           --  --  E37T                                                                              --  --  --  --  --  --                                      Important                                                                           --  --  --  --  --  --  K42E                                                                              --  --                                      Important                                                                           --  --  --  --  --  --  L44V                                                                              --  --                                      Important                                                                           W51A                                                                              W51A                                                                              --  --  W51A                                                                              W51A                                                                              --  W51A                                                                              --                                      Important                                                                           Y70 Y70 Y70 --  --  --  --  --  --                                      Important                                                                           --  --  --  --  Q75I                                                                              --  --  --  --                                      Minor K42E                                                                              L44V                                                                              K33I                                                                              --  K42E                                                                              --  L40A                                                                              --  --                                      Minor --  --  --  --  E32K                                                                              --  --  --  --                                      Minor --  --  W51A                                                                              --  --  --  --  --  --                                      __________________________________________________________________________

The mutations T38/A, S68/A and R64/Q had no effect on binding to any ofthe nine antibodies tested, while mutation E43/A resulted in 50%increase in binding of the above nine monoclonal antibodies incomparison to binding to wild type ICAM-R. Mutation F21V/AS abolishedbinding of all antibodies and appears to grossly affect the conformationof ICAM-R.

EXAMPLE 15

The distribution of ICAM-R protein and the expression of ICAM-R RNA invarious cells and cell lines were respectively assayed by FACS analysisand Northern blot hybridization.

A. FACS Analyses of ICAM-R Protein Distribution In Leukocytic Cell Linesand Normal Leukocytes

FACS analyses carried out as described in Example 12C on leukocyte celllines using anti-ICAM-R monoclonal antibody ICR-2. 1, anti-ICAM-1antibody (LB2) and anti-CD18 antibody (TS1/18, ATCC HB203) illustratedthat ICAM-R is expressed on a wide variety of in vitro propagated cellslines representative of the major leukocyte lineages: T lymphocytes, Blymphocytes, and myeloid cells. Surface expression of ICAM-R was notdetected on the primitive erythroleukemic line, K562. Further, ICAM-Rwas not expressed detectably by cultured human umbilical veinendothelial cells (HUVECS) either before or after stimulation with tumornecrosis factor which did upregulate expression of ICAM-1. This patternof expression is also distinct from that observed for ICAM-2 which isexpressed on endothelium. Table 7 below provides the mean fluorescenceof each cell sample and the percent positive cells relative to a controlin each cell sample (e.g., mean fluorescence of 13/11% positive cells).

                  TABLE 7                                                         ______________________________________                                        Cell Type                                                                              Cell Line  ICAM1     ICAMR  CD18                                     ______________________________________                                        T cell   CEM        13/11     212/99 160/99                                   T cell   MOLT4      ND        ND     15/77                                    T cell   HUT78      41/97     ND     110/99                                   T cell   SKW3       9/36      293/99 82/99                                    B cell   JY         ND        ND     60/99                                    B cell   JIJOYE     300/99    153/99 28/9                                     B cell   RAJI       229/99    98/96  51/98                                    Mono     HL-60      53/89     146/100                                                                              159/100                                  Mono     HL60-PMA   88/99     ND     251/100                                  Mono     U937       83/99     148/100                                                                              61/100                                   Mono     U937-PMA   68/100    ND     170/100                                  Myelo    KG-1       32/84     587/99 239/99                                   Myelo    KG-1a      32/90     238/97 83/93                                    Erythro  K562       37/0.84   31/0.27                                                                              ND                                       Endo     Huvec      51/18     57/1   ND                                       Endo     Huvec-TNF  278/99    36/1   ND                                       Human    Lymphocytes                                                                              31/19     388/99 305/99                                   Human    Monocytes  74/96     862/99 1603/99                                  Human    Granulocytes                                                                             12/40     323/99 376/99                                   Monkey   Lymphocytes                                                                              79/2      55/81  722/99                                   Monkey   Monocytes  98/1.7    162/95 1698/99                                  Monkey   Granulocytes                                                                             20/2      80/96  623/99                                   ______________________________________                                    

B. FACS Analyses of ICAM-R Distribution On Human and Macague Leukocytes

FACS analyses performed as described in Example 10C on normal human andmacaque peripheral blood leukocytes showed that the anti-ICAM-Rmonoclonal antibody ICR-2.1 reacted with the three major human leukocytelineages: lymphoid, monocytoid and granulocytoid. See the final sixentries of Table 7. In addition, monoclonal antibodies ICR-4.2 andICR-9.2 cross-reacted with macaque leukocytes (Table 2 and Example 11E)indicating that these monoclonal antibodies may be useful in monitoringthe expression of ICAM-R in disease models executed in this animal.

Human bronchiolar aveolar lavage cells (primarily macrophages) werestained with five anti-ICAM-R antibodies (ICR-2.1, ICR-3.1, ICR-4.2,ICR-6.2 and ICR-7.1) as described in Example 11E and analyzed by FACS.None of the antibodies specifically stained these cells. Other dataobtained via immunohistological tests suggests that ICAM-R can beexpressed on macrophages on interstitial spaces in the lung. (SeeExample 18, where the expression of ICAM-R in lung tissue is described.)

C. Northern Blot Analyses of ICAM-R RNA Expression in Leukocytic CellLines and HUVECS

RNA was extracted from human leukocyte cell lines and from HUVECS asdescribed in Example 6, and was analyzed by Northern blot hybridization(also as described in Example 6) by probing with either ICAM-R or ICAM-1cDNA. After phosphorimaging of the initial hybridization, blots werestripped and reanalyzed using a human actin probe. The results of theactin normalized Northerns of ICAM-R and ICAM-1 probed blots arepresented in FIG. 7(A through B) as bar graphs. At the RNA level, ICAM-Rwas expressed in a variety of leukocytic cell types. ICAM-R RNAexpression was not necessarily concomitant with the expression of ICAM-1RNA. For example, unstimulated HUVECS express low levels of ICAM-1 andexpression is

upregulated following TNF stimulation (FIG. 7B). In contrast, detectablelevels of ICAM-R message were not observed in unstimulated or stimulatedHUVECS (FIG. 7A).

EXAMPLE 16

The expression of ICAM-R transcript in endothelial cells was examined.Poly A+ mRNA was obtained from human skin angiomas and analyzed byNorthern blot using ICAM-R ³² P-labeled riboprobes to determine whethersplice variants of human ICAM-R were present in endothelial cells.

The angiomas analyzed were benign human vascular tumors. These surgicalsamples were frozen and first examined by immunohisto-chemistry, usingthe anti-ICAM-R monoclonal antibodies ICR-3.1 and ICR-4.2. Angiomas wereseparated into two groups based on the level of expression of ICAM-R,one group expressing high levels of ICAM-3, another group expressing lowor no detectable levels of ICAM-R. These benign tumors did not showsigns of inflammation and ICAM-R expression was almost completelyrestricted to endothelial cells.

For Northern blot analysis, other human tissues (cerebellum, liver, lungcarcinoma, abnormal small intestine and spleen) were used as controls.All tissue samples were frozen and stored at -80° C. PolyA mRNA wasextracted from angiomas and control tissue blocks using RNA STAT60 mRNAisolation reagents (Tel-test "B", Inc., Friendswood, Tex.). The tissuewas ground and homogenized, then total mRNA was extracted from thehomogenate with chloroform and precipitated. Poly A+ mRNA were purifiedfrom total mRNA by chromatography on oligo dT cellulose columns. Five ugof each polyA+ mRNA were loaded per lane on a 1% formaldehyde agarosegel, then transferred to Hybond-C membranes (as described in Example 7).

To generate human ICAM-R riboprobe, a subclone of pVZ-147 plasmid(Example 4) encoding ICAM-R domain 1 was utilized. The plasmid wasdigested with Asp718 and the complementary strand synthesized via T3primer and RNA polymerase using ³² P UTP. The resultant RNA transcriptwas used as a probe.

The nylon membranes were pre-hybridized and hybridized in 50% formamide,5× SSC, 1× PE (50 mM Tris-HCL pH 7.5, 0.1% sodium pyrophosphate, 0.2%polyvinylpyrolidone, 0.2% ficoll, 5 mM EDTA, 1% SDS) and 150 ug/mldenatured salmon sperm. RNA probes were denatured at 100° C. for 5minutes then added to each membrane at a concentration of 1×10⁶ dpm/mlhybridization mix. Membranes were hybridized overnight at 65° C., thenwashed at 65° C. twice in 2× SSC, 0.1% SDS and twice in 0.1× SSC, 0.1%SDS for 15 minutes each. They were exposed on film for three hours tothree days.

Analysis of the Northern blot demonstrated that the size of thetranscripts present in both the high and low expression groups ofangiomas was identical. Two transcripts were detectable, one migratingat about 2.2 kb identical to the one present in hematopoietic cells.Another transcript migrating at 3 kb was also present in both angiomasand control tissues that could correspond either to a variant of ICAM-Ror to cross-hybridization with another molecule. The level ofhybridization of both transcripts were high in the group of angiomasexpressing high levels of ICAM-R protein, while it was low in the lowexpressing group. The regulation of ICAM-R expression on endothelial andhematopoietic cells is apparently distinct; expression is constitutiveon hematopoietic cells, while it is induced on endothelial cells atneovascularizing sites. However, based on immunohistochemistry withanti-ICAM-R antibodies and on this Northern analysis, it seems likelythat a significant fraction of the ICAM-R molecules expressed onendothelial cells are likely to be identical in primary structure tothat of the molecule expressed on cells of hematopoietic origin.

EXAMPLE 17

Immunoprecipitations of detergent solubilized lysates of surfacebiotinylated human cell lines KG1a, K562 and CEM were performed usingthe four anti-ICAM-R monoclonal antibodies: ICR-2.1, ICR-1.1, ICR-4.2,and ICR-3.1.

Cell surface proteins on human leukocyte cell lines KG1, K562, and CEMwere labelled by reaction with sulfo-NHS-biotin (Pierce ChemicalCompany, Rockford, Ill.) as follows. For each reaction 0.5-1×10⁷ cellswere washed twice in phosphate buffered saline (PBS), resuspended in 1ml PBS and 10 μl of 100 mM sulfo-NHS-biotin diluted in PBS was added.Following incubation for 10 minutes at 37° C. the cells were washed oncewith PBS, and 4 ml of 10 mM Tris pH 8.4, 0.25M sucrose was added and thecells were then incubated for 30 minutes at 4° C. with gentle mixing.The cells were pelleted by centrifugation, the supernatant was aspiratedand the pellet was solubilized with 300 μl of 10 mM Tris pH 8, 50 mMNaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA byincubating on ice for 15 minutes. The lysate was clarified bycentrifugation and the supernatant was precleared by addition of 25 μlnormal mouse serum and incubation for 1 hour at 4° C. This step wasfollowed by the addition of 20 μl of a 50/50 (v/v) solution of protein-Asepharose beads (Sigma) that had been preincubated with 20 μg ofaffinity purified rabbit anti-mouse Immunoglobulin (Zymed). Afterincubation for 30 minutes at 4° C., the sepharose beads were removed bycentrifugation.

Specific immunoprecipitations were then performed by addition of 20 μlof sepharose beads that had been prearmed by sequential incubation withrabbit anti-mouse immunoglobulin and either anti-ICAM-R or control IgG₁or IgG_(2a) monoclonal antibodies. Following overnight incubation at 4°C. with agitation, sepharose beads were pelleted in a microcentrifugeand washed sequentially 2 times with 1 ml 10 mM Hepes pH 7.3, l150 mMNaCl, 1% Triton X-100; 1× with 0.1M Tris pH 8, 0.5M LiCl, 1% betamercaptoethanol; and 1× with 20 mM Tris pH 7.5, 50 mM NaCl, 0.5% NP-40.Beads were then eluted with 50 μl 150 mM Tris pH 6.8, bromphenol blue,20% beta mercaptoethanol, 4% SDS and 20% glycerol; boiled for 5 minutes;and pelleted by centrifugation. Thirty-five μl of the resulting eluatewas then analyzed by SDS-PAGE (10% acrylamide). After electrophoresis,proteins were electroblotted onto Immobilon-P membranes (Millipore,Bedford, Mass.) and incubated in 2% bovine serum albumin diluted inTris-buffered saline containing 0.2% Tween-20 for 20 minutes at 4° C.Blots were then incubated with horseradish peroxidase coupled tostreptavidin (Vector) in TBS-Tween at room temperature for 20 minutes.Following 3 rinses in TBS-Tween, ECL western blotting detection reagents(Amersham) were added and chemiluminescent bands were visualized onKodak X-OMAT-AR film.

FIG. 8(A through B) shows the resulting Western blots. A singlespecifically precipitated species of 120 kD was observed inimmunoprecipitates with monoclonal antibody ICR-2.1 from KG1 cells, butnot from K562 cells (See FIG. 8A).

A 120 kD band was also resolved in immunoprecipitates of the T cell lineCEM (FIG. 8B, wherein Lane A was reacted with monoclonal antibodyICR-2.1; Lane B, monoclonal antibody ICR-4.2; Lane C, monoclonalantibody ICR-3.1; Lane D, monoclonal antibody ICR-1.1; and Lane E, anegative control antibody). The size of the ICAM-R species resolved inother immunoprecipitations varied slightly depending on the cellularsource. Species ranging from about 116 kD on some lymphoid cells toabout 140 kD on some myeloid cells were observed. Given the predictedsize (about 52 kD) of the core peptide based on the nucleotide sequenceof the ICAM-R gene, these results imply that ICAM-R is heavily modifiedpost-translationally to yield the mature cell surface form of theprotein.

EXAMPLE 18

Immunohistologic staining with anti-ICAM-R monoclonal antibodiesICR-4.2, ICR-1.1, and ICR-2.1 and control antibodies was carried out onvarious human tissues including tonsil, spleen, liver, lung, kidney,heart, digestive tract, skin, synovium, and brain (both normal andmultiple sclerosis-afflicted brain tissue). Similar staining patternswere obtained using the different anti-ICAM-R antibodies as well as whenusing purified anti-ICAM-R monoclonal antibody ICR-1.1 or hybridomasupernatant.

Sections (6 μm) of various tissues were layered onto Vectabond (Vector)coated slides and stored at -70° C. (some sections were stored at -20°C.). Prior to use, slides were removed from -70° C. and placed at 55° C.for 5 minutes. Sections were then fixed in cold acetone for 10 minutesand air dried. Sections were blocked in a solution containing 1% BSA,60% normal human sera, and 6% normal horse sera for 30 minutes at roomtemperature. Primary antibody directed against ICAM-R, a negativecontrol antibody, anti-ICAM-1 monoclonal antibody or anti-ICAM-2monoclonal antibody was applied to each section for 1 hour at roomtemperature. Unbound antibody was washed off by immersing the slides 3times in 1× PBST for 5 minutes each time. Biotinylated anti-mouseimmunoglobulin (Vector) was then applied to each section in the samefashion. ABC-HPO (Avidin-Biotin Complex-HPO) was used to detect thesecond antibody. A solution of reagent A (9 μl) (Vector) combined withreagent B (9 μl) (Vector) in 1 ml of 1% BSA/PBST was applied to eachsection for 30 minutes at room temperature. Slides were then washed 3times in 1× PBST. DAB substrate (3'3diaminobenzidine-tetrahydrochloride, Sigma) (stock: 600 mg/ml DABdiluted 1:10 in 0.05M Tris Buffer, pH 7.6, with 3% H₂ O₂ added to afinal concentration of 1%) was applied to each slide for 8 minutes atroom temperature. Slides were washed in water for 5-10 minutes at roomtemperature and then 1% osmic acid was added (to enhance colordevelopment) for one minute at room temperature. Slides were then washedin tap water for 5-10 minutes and counterstained in 1% Nuclear Fast Red(NFR) for 30 seconds at room temperature. Lastly, slides were alcoholdehydrated, treated with Histroclear and mounted with coverslips usinghistomount.

A selection of results of staining with the monoclonal antibodies ispresented in FIG. 9(A through G) as photomicrographs wherein the tissuein 9A, 9B and 9E is human tonsil; in 9C and 9D is human liver; in 9F isbrain from a human patient afflicted with multiple sclerosis; and in 9Gis normal human brain. Sections shown in 9A, 9C, 9F and 9G were stainedwith anti-ICAM-R monoclonal antibody ICR-4.2. Sections shown in 9B and9D were stained with the negative control antibody, while the sectionshown in 9E was stained with the anti-ICAM-1 antibody. Staining revealedhigh level expression of ICAM-R in lymphoid tissues such as tonsil (9A).Expression was also detected on tissue leukocytes in other nonlymphoidorgans such as the liver wherein Kupfer cells (liver macrophages) werepositively stained (9C). Evidence that ICAM-1 and ICAM-R expression areregulated distinctly in vivo is given by the staining pattern observedin tonsil and lymph node: ICAM-1 is strongly expressed on B cells in thegerminal centers of secondary follicles and not expressed in primaryfollicles, whereas ICAM-R is expressed strongly in the primary folliclesand weakly in the germinal centers (10A and 10E). Significantly, ICAM-Rexpression was also detected on leukocytes infiltrating sites ofinflammation. For example, ICAM-R expression was observed onperivascular infiltrating leukocytes in the brain tissue of individualsafflicted with multiple sclerosis (9F). Similar staining was notobserved in anatomically equivalent locations of brain tissue fromnormal individuals (9G). ICAM-R expression was also detected onleukocytes infiltrating synovia of arthritic joints. Also, whereasexpression of ICAM-1 and ICAM-2 was detected on endothelia liningvessels, ICAM-R was not typically observed on vascular endothelium.Expression of ICAM-R was detected on cells in the aveoli of the lung.

More generally, cells expressing ICAM-R were detected in all normal andpathological tissues. These ICAM-R expressing cells could be identifiedmorphologically and by comparison of serial immunological staining asleucocytes and antigen-presenting cells. All CD3⁺ T cells present invarious tissues expressed high levels of ICAM-R. In contrast, only asubset of B cells (IgD+) present in primary follicles and in the mantlezone of germinal centers expressed high levels of ICAM-R. Amongstantigen-presenting cells, Langerhans cells in the epithelium expressedhigh levels of ICAM-R while only a subset of other tissue macrophagesexpressed ICAM-R.

ICAM-R monoclonal antibodies ICR-1.1 and ICR-4.2 were also used inprocedures similar to those described above to stain biopsy tissuesections of both human mammary carcinoma (ductal and lobular) andmelanomas. In both tumor types some sections exhibited specific patchystaining of the endothelia in a range of blood vessels (venular,arterioles and capillaries). Corresponding normal tissue showed noexpression of ICAM-R on endothelium.

Thus, while ICAM-R is typically not expressed on endothelium of thegeneral vasculature, it is apparently expressed on a subset of vesselsassociated with two types of solid tumors. Given this distribution,reagents (e.g., monoclonal antibodies) directed against ICAM-R mayprovide therapeutic vehicles which selectively target tumor versusnormal vasculature.

In summary, the contrasts in the patterns of expression of ICAM-R versusICAM-1 and ICAM-2 are significant. Constitutive expression of ICAM-2 wasobserved on both leukocytes and endothelium. Basal expression of ICAM-1on leukocytes, endothelia and epithelia was low or absent but wasinduced in pathologic tissues or in vitro. ICAM-R was expressed at highlevels on most leukocytes and, notwithstanding rare expression on tumorassociated endothelia, was generally not expressed on vascularendothelia.

Since macrophages are involved in cholesterol deposition and generationof atherosclerotic lesions, both thoracic aorta and abdominal aortasections from PDAY (Pathological Determinants of Atherosclerosis inYouth, LSU Medical Center) tissue samples were analyzed with anti-ICAM-Rand anti-alpha d antibodies. The lesions examined were consistent withaortic fatty streaks consisting of subintimal aggregates of large foamcells (mostly macrophages with ingested lipis and infiltrates of smallerleukocytes.

Double label studies were conducted to determine the relativelocalization of ICAM-R and alpha d antigens in the aortic sections.Small ICAM-R positive leukocytes were surrounded by and interspersedwith CD68 positive macrophages expressing alpha d. There was a limitednumber of small leukocytes which were CD68 negative, but stained withboth ICAM-R and alpha d specific antibodies.

The apposed distribution of ICAM-R and alpha d positive cells, supportedby evidence suggesting an interaction between this CAM/integrin pair(Example 26), suggests that the functional consequences of ICAM-Rengagement by alpha d may lead to events that are important drivingforces in the pathology of atherosclerosis. Antibodies to the inventionhave been shown to elicit chemokine release, particularly MCP-1 frommonocytes. MCP-1 has been shown to be localized to macrophages inatherosclerotic lesions. Taken together, these results imply that alphad engagement of ICAM-r signals macrophages to release MCP-1.

EXAMPLE 19

In order to determine whether ICAM-R is involved in homotypic celladhesion, aggregation assays were performed with a panel of cell lineswhich express ICAM-R including T lymphoblastoid cell lines (SupT1, CEM,Molt 4, Hut 78, Jurkat, SKW3), B lymphoblastoid cells lines (Jijoye,Raji), monocytic cell lines (U937, HL60), a myelogenous cell line (KG-1)and the erythroleukemia cell line K562. To determine the function of theICAM-R molecule, the cells were incubated with various antibodies beforeaggregation was assayed. Anti-ICAM-R supernatants produced by hybridomasICR-2.1, ICR-1.1, ICR-4.2, and ICR-3.1 were used as well as antibodypreparations known to block aggregation through a β2 integrin pathway:TS1/18 (ATCC HB203) specific for the CD18 molecule, the β-subunit ofLFA-1; TS1/22 (ATCC HB202) specific for the CD11a molecule, the α-chainof LFA-1; and LM2/1 (ATCC HB204) specific for the CD11b molecule, theα-subunit of MAC-1. Purified anti-ICAM-1 antibody and hybridomasupernatant directed against the α-chain of the VLA-4 molecule(hybridoma clone 163H, Michael Longenecker, Alberta, Canada) were usedas controls.

Aggregation assays were done in duplicate, with and without addition ofPMA (50 ng/ml). 3×10⁵ cells in RPMI 1640 medium with 10% fetal calfserum were added in a flat-bottomed 96-well microtest plate. When oneantibody was tested in an experiment, 50 μl of purified antibody orhybridoma supernatant were added to the wells (PMA was added at the sametime to selected wells). When two antibodies were tested in the sameexperiment, the antibodies were added sequentially to the cells at roomtemperature and incubated for 30 minutes each (incubation for 15 minutesat 37° C. produced the same results), and then the cells were incubatedat 37° C. Incubating the antibodies with the cells before addition ofPMA or at the same time as the PMA did not cause any significant changein the aggregation results. After incubation with the antibody orantibodies, cells were uniformly resuspended and then incubated at 37°C. for 4 to 24 hours. Aggregation scoring was done with an invertedmicroscope. In each experiment, the efficacy of the PMA stimulation waschecked in parallel by stimulating Raji cells with an equal amount ofPMA and determining the amount of aggregation blockable by monoclonalantibodies to CD18, CD11a, and ICAM-1 molecules.

Table 8, below, sets out the results of one representative aggregationexperiment wherein PMA was added. Aggregation scores are reported on arange from 0 to 5, wherein 0 indicates that no cells were in clusters; 1indicates that less than 10% of the cells were in clusters; 2 indicatesthat 10 to 50% cells were aggregated; 3 indicates that 50 to 100% cellswere in loose clusters; and 4 indicates that almost 100% of the cellswere in compact aggregates.

                                      TABLE 8                                     __________________________________________________________________________    Antibody Treatment                                                            Antibody 1                                                                             --                                                                              --  --   --   --  --  αCD18                                                                       αCD11a                                                                       αCD11b                        Antibody 2                                                                             --                                                                              αCD18                                                                       αCD11a                                                                       αCD11b                                                                       26H11C                                                                            26I10E                                                                            26H11C                                                                            26H11C                                                                             26H11C                              Aggregation                                                                   SUPT1 cells                                                                            2 1   1    2    4   2   2   2    4                                   (after 4 hours)                                                               SUPT1 cells                                                                            2 1   1    2    4   2   2   2    4                                   (after 24 hours)                                                              __________________________________________________________________________

Interestingly, treatment with three of the antibodies specific forICAM-R (ICR-2.1, ICR-1.1, and ICR-3.1) stimulated homotypic cell-cellaggregation (data for ICR-1.1 and ICR-3.1 not shown). Stimulationoccurred in both the presence and absence of co-stimulatory agents suchas a phorbol ester (PMA). The fourth anti-ICAM-R monoclonal antibody(ICR-4.2) did not stimulate cell aggregation but blocked the aggregationstimulated by the other anti-ICAM-R antibodies. At least a portion ofthe aggregation stimulated by anti-ICAM-R antibodies in PMA treatedcells was blocked by pretreatment with monoclonal antibodies againstCD18 or CD11a indicating that one or more leukointegrins may participatein this type of adhesion.

To confirm that aggregation was induced by the anti-ICAM-R antibodiesICR-2.1, ICR- 1.1, and ICR-3.1 the aggregation assays were performedusing both the whole immunoglobulin (ICR-1.1-Ig) and Fab' fragments(ICR-1.1-Fab') purified from the same anti-ICAM-R monoclonal antibody(ICR-1). The assays were performed with SKW3 T cells as described aboveusing ICR-1.1-Ig and ICR-1.1-Fab' at a concentration of 1 μg/ml.Supernatants of anti-CD18 and anti-ICAM-R (CIR-1.1-sup and ICR-4.2-sup)hybridomas were used as controls. After four hours, the same increase incell aggregation was found for whole immunoglobulin as for the Fab'fragments or the ICR-1.1 supernatant (See Table 9 below).

                                      TABLE 9                                     __________________________________________________________________________    Antibody Treatment                                                                     0 αCD18                                                                       26E3D-Ig                                                                           26E3D-Fab'                                                                          26E3d-sup                                                                          26I10E-sup                                     Aggregation                                                                            2 2   3    3     3    2                                              __________________________________________________________________________

No increase in aggregation was found with anti-CD18 supernatant oranti-ICAM-R ICR-4.2 supernatant. These results rule out the trivialexplanation that enhanced aggregation was due to antibody mediatedcross-linking of the cells. The engagement of ICAM-R protein, in thiscase by selected antibodies, may transduce a signal which alters theadhesive potential of the bound cells.

EXAMPLE 20

The process of activation and proliferation of cells of the immunesystem is marked by a continuum of cellular events. The upregulation ofcertain cell surface molecules (e.g., CD69 and the transferrin receptor)is an early marker of cell activation. Similarly, cell agglutinationoccurs early in the process of activation. The upregulation of the IL-2receptor occurs at an intermediate to late stage and cell proliferationis a late event. Six types of experiments were performed to determinethe extent to which ICAM-R is involved in immune cellactivation/proliferation. In the first type, the capacity of ICAM-Rpresented on the surface of a transfected cell to stimulateproliferation of lymphocytes was examined. In the second type,antibodies of the invention recognizing distinct epitopes on ICAM-R wereused as probes to engage the external domain of ICAM-R to determine theeffects of antibody binding either alone or in combination with otherstimuli on lymphocyte or monocyte activation and proliferation. In thethird type of experiment, the effects of recombinant ICAM-R protein on Tcell proliferation were determined. In the fourth type, variant ICAM-Rproteins were expressed in lymphoblastoid cells and effects of themutations on T cell receptor-dependent stimulation were measured. In thefifth type of experiment, the downstream intracellular biochemicalconsequences (e.g., effects on PKC translocation) of ICAM-R engagementwere examined. In the sixth type, the effects of antibodies of theinvention on release of acute basophil mediators was determined.

A. Stimulation of PBMC Proliferation by ICAM-R Transfectants

Mouse L cells transfected with either ICAM-R cDNA or ICAM-1 cDNA(Example 7) were assayed for their ability to stimulate human peripheralblood mononuclear cell (PBMC) proliferation as measured by ³ H-thymidineincorporation assays which indicate changes in the rate of DNAreplication. Nontransfected mouse L cells or transfected L cells wereobtained by trypsinization from tissue culture flasks and washed inRPMI-1640 containing 10% fetal bovine serum. Five×10⁴ L cells in 120 μltissue culture media (RPMI-1640 with 10% fetal bovine serum) were addedto individual wells of a sterile 96-well flat bottom tissue cultureplate and the plates were incubated for 24-36 hours at 37° C. in a 5%CO₂ incubator. The media was then removed in a sterile manner and 2×10⁵freshly isolated PBMC in a total volume of 200 μl tissue culture mediawere added to individual wells containing either transfected ornon-transfected mouse L cells. PBMC were also added to control wellscontaining no L cells. The PBMC were previously isolated from healthydonors by centrifugation on Histopaque gradients (Sigma). Freshperipheral blood was mixed with an equal volume of PBS, layered ontoHistopaque and centrifuged at 450 g for 20 minutes with no brakeapplied. PBMC-containing fractions were collected, washed in PBS andadjusted to 1×10⁶ viable cells/ml prior to addition into wells. Thetissue culture plates were then incubated for a total of 4 days eitherin the presence or absence of PMA at a final concentration of 5 ng/ml.Lymphocyte proliferation was then assessed after the addition of 1 uCi ³H-thymidine (NEN, Boston, Mass.) to individual wells for the last 18-24hours of culture. All cultures were then terminated by harvesting thecontents of each well onto glass fiber filter strips using a PHD modelplate harvester (Costar, Cambridge, Mass.). Individual filter mats werethen placed in 3 ml Ecolume scintillation cocktail (ICN Biomedicals,Costa Mesa, Calif.) and counted using a beta-scintillation counter. LTKcells expressing ICAM-R stimulated proliferation of PBMC (as indicatedby increased DNA replication) in comparison to nontransfected controlLTK cells or in the absence of any stimulus. LTK cells expressing ICAM-1induced the proliferation of PBMC to approximately an equal extent. Bybinding to its receptor(s) on PBMC, ICAM-R transmits an intercellularsignal to the PBMC which in this cellular context results in cellproliferation.

B. PMBC Activation by ICAM-R Specific Monoclonal Antibodies

Anti-ICAM-R antibodies of the invention were also tested to determinetheir effect on immune cell activation and proliferation.

Anti-ICAM-R monoclonal antibodies were preliminarily tested for theability to affect early events in cell activation including upregulationof the cell surface molecules CD69, the transferrin receptor and theIL-2 receptor on the target cells as measured by flow cytometryanalysis. Unstimulated lymphocytes express low levels of the transferrinand IL-2 receptors. Expression of the receptors increases dramaticallywhen lymphocytes are activated.

Anti-ICAM-R monoclonal antibodies ICR-1.1 and ICR-4.2 were each testedfor the ability to induce PMBC activation in the absence of otherinducing stimuli. Monoclonal antibodies ICR-1.1 or ICR-4.2 (or controlmonoclonal antibodies) were added (10 μg/well in PBS) to individualwells of a 96-well flat bottom tissue culture plate and incubated for 3hours at 37° C. in a 5% CO₂ incubator. The plates were washed 3 timeswith sterile PBS to remove unbound antibody and freshly isolated PBMCwere immediately added to a final concentration of 2×10⁵ cells/well in avolume of 200 μl media. The plates were then incubated for either 1 or 3days at which time the cells cultured in the presence of differentantibodies were removed, washed as described above in PBS containing0.01% sodium azide and 1% BSA (FACS buffer) and stained with either FITC(Becton Dickinson) -conjugated negative control antibodies or a panel ofFITC-conjugated anti-CD69, anti-transferrin receptor and anti-IL-2receptor antibodies. Results were obtained by FACScan analysis.Expression of CD69 and the transferrin receptor but not the IL-2receptor increased after 1 day when PBMC were cultured on immobilized(i.e., cross-linked) antibody ICR-1.1 but not when cultured onimmobilized antibody ICR-4.2 PBMC incubated for 3 days on immobilizedICR-1.1 or ICR-4.2 had increased levels of cell surface expression ofboth the transferrin receptor and IL-2 receptor but not CD69. However,while increased expression of these lymphocyte activation markers wasobserved after 1 and 3 days this increased expression was unaccompaniedby increased cell size. These results suggest that the anti-ICAM-Rmonoclonals ICR-1.1 and ICR-4.2 are able to directly induce early eventsin PMBC activation in the absence of additional exogenous stimuli butthis activation does not result in blast transformation and associatedincreases in cell size.

C. Effect of ICAM-R Specific Monoclonal Antibodies on Stimulation ofPMBC Activation by Anti-CD3 Antibody

Anti-ICAM-R monoclonal antibodies were also tested for their ability toalter early events in PMBC activation stimulated by immobilized anti-CD3monoclonal antibody G19 Ledbetter et al., J. Immunol., 135(4): 2331-2336(1985)!. Monoclonal antibody G19 binds to the CD3 complex on T cells(the T cell receptor) and activates T cells. When PBMC were cultured inwells precoated with anti-CD3 antibody (0.05 μg/well) alone, only CD69expression was elevated after one day. After three days, cell surfaceexpression of CD69, the transferrin receptor and the IL-2 receptor wasdramatically elevated. Upregulation of these activation markers wascorrelated with increases in cell size.

Ten μg of anti-ICAM-R monoclonal antibodies ICR-1.1 or ICR-4.2 (orcontrol monoclonal antibodies to HLA Class I; Serotec, Oxford, England)were added per well of 96-well flat bottom tissue culture plates eitherin the presence or absence of anti-CD3 antibody initially added at 0.025μg/well and washed to remove unbound antibody. Freshly obtained PBMCwere immediately added (2×10⁵ cells/well). The cells were then incubatedfor a total of either 16 hours or 3 days at which time the cells wereremoved and washed 2 times in ice cold FACS buffer. Two×10⁵ cells werethen resuspended in 50 μl ice cold FACS buffer, and 5 μl ofFITC-conjugated anti-CD69, anti-transferrin receptor, anti-IL-2 receptorantibody or anti-FITC conjugated control Ig was added. The cells wereincubated at 4° C. for 30 minutes and then washed 2 times in 0.5 ml icecold FACS buffer. After the final wash the cells were resuspended in 0.5ml FACS buffer and fluorescence determined by FACScan analysis. WhenPBMC were cultured for 3 days on 0.025 μg/well immobilized anti-CD3either alone or in the presence of immobilized antibody to HLA Class I,expression of the transferrin and IL-2 receptors is not upregulated atthis low does of immobilized anti-CD3. In contrast, culturing of PBMC inthe presence of 0.025 μg/well immunobilized anti-CD3 and eitherimmobilized anti-ICAM-R antibodies ICR-1.1 or ICR-4.2 antibodiesresulted in significant upregulation of both the transferrin and IL-2receptors. The effect was more pronounced with antibody ICR-1.1. Similarresults were also obtained after 16 hours in culture. Low dose anti-CD3in the presence of immobilized ICR-1.1 or ICR-4.2 antibody inducedexpression of CD69, but not the transferrin receptor, while low doseanti-CD3 (0.025 μg/well) in the presence of immobilized anti-HLA-I didnot induce increased expression of either CD69 or the transferrinreceptor. These results indicate that these anti-ICAM-R antibodies mayserve as costimulatory molecules in early immune cell activation events.

D. Stimulation of PMBC Proliferation in the Presence of IL-2

Preliminary experiments were performed to determine if anti-ICAM-Rmonoclonal antibodies could affect the late event of cell proliferationagain as measured by ³ H-thymidine incorporation assays.

Monoclonal antibodies to ICAM-R were tested for their ability todirectly stimulate PMBC proliferation in either the presence or absenceof human recombinant IL-2 which potentiates but does not induce cellproliferation. Ten μg of ICAM-R monoclonal antibodies ICR-1.1 or ICR-4.2(or control IgG₁ and IgG₂) antibodies) in PBS were added per well of96-well flat bottom tissue culture plates and the plates were incubatedfor 3-4 hours at 37° C. in a 5% CO₂ incubator. After incubation, eachwell was rinsed 3 times with PBS and freshly obtained PBL were added toa final concentration of 2×10⁵ cells/well in a volume of 200 μl. Tenunits/ml human recombinant IL-2 (Genzyme, Boston, MA) was then added toselected wells. The plates were incubated for a total of 3 days at 37°C. in a 5% CO₂ incubator. ³ H-thymidine incorporation by the PMBC wasdetermined as described earlier in this example. The anti-ICAM-Rantibodies ICR-1.1 and ICR-4.2 did not induce PMBC proliferation even inthe presence of rIL2. Positive controls for lymphocyte proliferationincluded immobilized anti-CD3 and anti-LFA-1 (60.3) monclonalantibodies. These results indicate that while the immobilizedanti-ICAM-R antibodies stimulate expression of activation markers suchas CD69, etc., by themselves they do not directly stimulate the entry oflarge numbers of PBMC into S phase of the cell cycle.

E. Costimulation of Lymphocyte Proliferation by ICAM-R SpecificAntibodies

Because anti-ICAM-R antibodies with anti-CD3 antibodies costimulatedearly PBMC activation events, anti-ICAM-R antibodies were tested for theability to costimulate lymphocyte proliferation induced by immobilizedanti-CD3 antibody. In addition, to determine whether anti-ICAM-Rantibodies costimulate T-lymphocytes in the absence of accessory cells,anti-ICAM-R antibodies were tested for their ability to costimulateproliferation of pure CD4⁺ T-lymphocytes, isolated using negativeselection. To isolate CD4⁺ cells PBMC were suspended in tissue culturemedium, added to 75 ml tissue culture flasks (Corning) and incubated for1 hour at 37° C., 5% CO₂. Plastic nonadherent cells were then removedfrom the flask by gently rinsing once with PBS. The nonadherent cellfraction was suspended (10⁷ cells/ml) in an antibody cocktail containing1 μg/ml anti-CD8 antibody (Pharmingen, San Diego, Calif.), 1 μg/mlanti-CD19 (Becton Dickinson), 1 μg/ml anti-CD11b (Becton Dickinson) in10% FBS-PBS (coating medium), and incubated for 1 hour at 4° C. Unboundantibody was removed by washing twice in coating medium. Cells were thenresuspended (10⁷ cells/ml) in coating medium containing Goat-anti-mouseIg coated magnetic beads (45 μl/10⁶ cells)(Advanced Magnetics,Cambridge, Mass.) and incubated for 1 hour at 4° C. Cells bound tomagnetic beads were then removed from suspension using a strong magnet.CD4⁺ populations obtained using this method were found to be >90% pureby flow cytometric analysis. PBMC or CD4⁺ cells were adjusted to aconcentration of 1×10⁶ viable cells/ml in tissue culture medium.Individual wells of a 96-well flat bottom tissue culture plate wereprecoated with 0.001 μg anti-CD3 monoclonal antibody G19 per well. Theplates were incubated for 3 hours at 37° C. in a 5% CO₂ incubator andunbound antibody was removed by rinsing the wells 3 times in PBS. Afterthe final PBS wash, monoclonal antibodies to ICAM-R (ICR-4.2 or ICR-1.1)or control antibodies were immediately added to a final concentration of10 μg/well. The plates were then reincubated for an additional 3 hoursat 37° C. The wells were again washed three times with PBS to removeunbound antibody and freshly isolated PBMC were immediately added to thewells (2×10⁵ cells in a volume of 200 μl/well). The plates were thenincubated for 3 days. Lymphocyte proliferation was measured by ³H-thymidine incorporation by the PMBC or CD4⁺ cells. As shown in FIG. 10immobilized anti-ICAM-R monoclonal antibodies ICR-1.1 and ICR-4.2increased the PBMC and purified CD4⁺ cell response to anti-CD3. Effectsof the immobilized anti-ICAM-R antibodies on PBMC aggregation (anearlier event than PBMC proliferation) induced by anti-CD3 monoclonalantibody were also examined in this experiment. Anti-CD3 stimulatedaggregation was inhibited almost 100% by antibody ICR-1.1 but wasunaffected by immobilized ICR-4.2 and minimally inhibited by antibodiesICR-2.1 and ICR-4.1.

The results of the assays for the ability of anti-ICAM-R antibodies toaffect the proliferation of cells on which ICAM-R is expressed indicatethat binding of the antibodies of the invention to ICAM-R transmits adirect intracellular signal to T lymphocytes which modulates cellproliferation.

F. Co-Stimulation of Lymphocytes by Soluble ICAM-R

shICAM-R (Example 9) was assayed for the ability to costimulate humanlymphocyte activation. Human peripheral blood lymphocytes (PBL) wereobtained by Ficoll-Hypaque centrifugation and 2×10⁵ cells per well wereincubated in the presence of either media, plate bound shICAM-R, platebound anti-CD3 (OKT3) or a combination of plate bound anti-CD3 andshICAM-R. At 17 hours and 4 days after initiation of culture cells wereremoved, stained with monoclonal antibodies to human lymphocyteactivation antigens and analyzed by flow cytometry.

Human lymphocytes cultured for 4 days in the presence of plate boundanti-CD3 (0.5 ug/well) and shICAM-R (100 ng/well) express elevatedlevels of the activation antigens ICAM-1, IL-2 receptor and transferrinreceptor compared to lymphocytes cultured in the presence of anti-CD3alone. In contrast, lymphocytes cultured in the presence of solubleICAM-R (100 ng/well) alone expressed no increased levels of theseactivation antigens compared to cells cultured in media alone.

Experiments were also performed to determine if ICAM-R is involved inearly events of qualitatively distinct types of cell-cell contactdependent T-lymphocyte activation (e.g., responses to staph enterotoxinA and alloantigen).

G. Effect of ICAM-R Specific Antibodies on Superantigen-InducedProliferation of PBL

Superantigen-induced proliferation and aggregation of human PBL wereassessed in the presence of the ICAM-R specific antibodies of theinvention. The effect of soluble and plate-bound anti-ICAM-R antibodiesand anti-HLA class I control B-H9 (Serotec) antibodies on proliferationand cell aggregation was measured three days after stimulation of humanPBL with Staphylococcus Enterotoxin A (SEA) (Toxin Technology, Sarasota,Fla.). Plate-bound antibodies were prepared on the day of culture asfollows. Purified antibody (10 μg in 0.1 ml PBS) was added to individualwells of 96-well flat bottom plates. Plates were then incubated for 4hours at 37° C. Following incubation, unbound antibody was removed byaspirating each well and rinsing 4 times with fresh PBS. Human PBL wereisolated from healthy donors on Histopaque (Sigma) gradients. Freshperipheral blood was mixed with an equal volume of phosphate bufferedsaline (PBS), layered onto Histopaque and centrifuged at 450×g for 20minutes with no brake applied. Lymphocyte fractions were collected andwashed twice by adding a fresh volume of RPMI supplemented with 10%fetal bovine serum and centrifuging at 200×g for 8 minutes. PBL weresuspended in a final volume of 10 ml of RPMI-FBS. Viable PBL werecounted using the method of vital dye exclusion. Twenty μl of a dilutionof cell suspension in 0.4% trypan blue stain (Gibco) was added to ahemacytometer chamber and dye-excluding cells were then counted using aninverted microscope. Two-hundred thousand viable PBL were then added to96-well flat-bottom tissue culture plates containing 100, 10 or 1 μgsoluble or plate-bound ICR-1.1 ICR-2.1, ICR-3.1, ICR-4.2, ICR-5.1,ICR-6.2, ICR-7.1, ICR-8.1, ICR-9.2, ICR-12.1, ICR-13.1, ICR-14.1,ICR-15.1, ICR-16.1, ICR-17.1, B-H9 or IOT2 (AMAC, Inc., Westbrooke, Me.)antibodies. Finally, each culture was stimulated with SEA (1000 or 10pg/ml in triplicate) and cultured at 37° C. in 5% CO₂. After 3 days,proliferation was measured as ³ H-thymidine incorporation.

Treatment with soluble anti-ICAM-R antibodies failed to alterproliferation in comparison to soluble control antibodies. Plate-bound(i. e., cross-linked) antibodies ICR-1.1, ICR-2.1, ICR-5.1, ICR-6.2ICR-8.1 and ICR-17.1 however, significantly inhibited proliferation inresponse to SEA (p<0.05) while antibodies ICR-3.1, ICR-4.2, ICR-7.1,ICR-9.2, ICR-13.1, ICR-14.1 and ICR-15.1 did not (FIG. 11A and FIG.11B). Antibodies ICR-12.1 and ICR-16.1 inhibited proliferation slightly,while antibodies ICR-12.1, ICR-13.1, ICR-14.1, ICR-15.1 and ICR-16.1exhibited enhancing effects at the lowest concentration. AntibodiesICR-1.1 and ICR-8.1 were the most effective at inhibiting proliferation.FIG. 11C presents logistic dose response curves for monoclonalantibodies ICR-1.1, ICR-2.1, ICR-5.1, ICR-6.2 and ICR-8.1 in terms ofthe percentage of proliferation observed compared to proliferation inthe presence of control antibodies and Table 10 below sets out the IC₅₀values obtained from the curves.

                  TABLE 10                                                        ______________________________________                                        Monoclonal Antibody   IC.sub.50 (μg/ml)                                    ______________________________________                                        ICR-1.1               63                                                      ICR-2.1               1434                                                    ICR-5.1               170                                                     ICR-6.2               80                                                      ICR-8.1               1                                                       ______________________________________                                    

Concomitant to inducing entry into the cell cycle, SEA induces cellaggregation. Effects of the monoclonal antibodies ICR-1.1 and ICR-4.2 oncell aggregation were measured using an inverted microscope. Plate-boundICR-1.1 also significantly inhibited cell aggregation at both SEAconcentrations in comparison to plate-bound B-H9 and ICR-4.2 antibodies.Inhibition of aggregation by plate-bound ICR-1.1 was almost complete. Incontrast, plate-bound ICR-4.2 antibody only slightly inhibitedaggregation in comparison to plate-bound B-H9. Aggregation of PBLinduced by SEA was not affected by soluble anti-ICAM-R antibodiesICR-1.1 or ICR-4.2 in comparison to soluble B-H9 antibody.

The minimum time required for plate-bound anti-ICAM-R to inhibitSEA-induced proliferation was also determined. PBL were pre-incubated onplate-bound ICR-4.2, ICR-1.1 or isotype-matched anti-HLA-I controlantibodies B-H9 (IgG₁) and IOT2 (IgG₂) with or without SEA (10 pg/ml)for 3, 5 and 7 hours. PBL were then transferred to clean wells andcultured in the presence of SEA (10 pg/ml) for 3 days. The results of ³H-thymidine incorporation (proliferation) assays are summarized in FIG.12. Immobilized ICR-1.1 antibody and, to a lesser extent ICR-4.2antibody, significantly reduced proliferation in comparison toisotype-matched controls after only 3 hours of incubation. This resultindicates that binding of plate-bound ICR-1.1 or ICR-4.2 to ICAM-Rtransmits an intracellular signal capable of inhibiting proliferationeven after cells have been removed from the immobilized antibodies.These results suggest that therapeutically efficacious engagement ofICAM-R may be achieved without maintaining saturating levels of anICAM-R specific agent (e.g., a monoclonal antibody) over long periods oftime.

Because both T cells and accessory cells express high levels of ICAM-R,the inhibition of cell-cell contact dependent T cell activation duringthe response to SEA by ICR-1.1 could be mediated by ICR-1.1 binding to Tcells, accessory cells or both. Additionally, because ICAM-R and ICAM-1differ markedly in their expression on nonactivated T cells, it ispossible that anti-ICAM-1 and anti-ICAM-R may inhibit the SEA responseby targeting T cell subsets in different states of activation. Becausethe role of ICAM-R may differ in naive and memory cells, the ability ofanti-ICAM-R antibodies to inhibit SEA induced proliferation of CD4⁺CD45RO⁺ ("memory") cells, or CD4⁺ CD45RA⁺ ("resting") cells was tested.Plasmatic nonadherent PBMC (10⁷ cells/ml) were incubated for 1 hour at4° C. with a cocktail of antibodies (1 μg/ml each) containing anti-CD8,anti-CD19, anti-CD11b, anti-HLA-DR (Becton Dickinson) and eitheranti-CD45RO (Amac) (to obtain CD45RA⁺ CD4⁺ cells), or anti-CD45RA (Amac)(to obtain CD45RO⁺ CD4⁺ cells) in coating medium. The cell suspensionwas washed twice with coating medium to remove unbound antibody andincubated with goat anti-mouse IgG coated magnetic beads. Cells bound tomagnetic beads were then removed from the suspension using a strongmagnet. CD45RO⁺ and CD45RA⁺ populations obtained using this method werefound to be >95% pure as determined by flow cytometric analysis. Twohundred thousand purified memory T cells, resting T cells or plasticadherent cells were incubated on immobilized ICR-1.1, anti-ICAM-1antibody LB-2 or anti-HLA-I antibody p10.1 (10 μg/ml) (Gerald Nepom,Virginia Mason Research Center, Seattle, WA) for 3 hours. The antibodytreated memory or resting T cells were removed to clean wells andadmixed with 2×10⁴ plastic adherent cells. Antibody treated accessorycells were admixed with either untreated memory T cells or untreatedresting T-cells. Each reconstituted culture was then stimulated with SEA(10 pg/ml). The results of ³ H-thymidine incorporation (proliferation)assays are summarized in FIG. 13 wherein the abbreviation "APC" standsfor "antigen presenting cells," which are the accessory cells in thisassay, and wherein the asterisks indicate the population of cellspretreated with antibody. Pretreatment of CD45RO⁺ T cells or accessorycells with ICR-1.1 blocked proliferative responses to SEA in comparisonto p10.1 control antibody. When both cell populations were treated withICR-1.1, the inhibitory effect was additive. Inhibition of proliferationby the anti-ICAM-1 antibody LB-2, occurred only when adherent cells werepretreated and was not further enhanced when the admixed cells were alsopretreated. As shown in FIG. 14 pretreatment of CD45RA⁺ T cells withICR-1.1 did not affect SEA responses. ICR-1.1 or LB2 pretreatment ofadherent cells resulted in modest inhibition of CD45RA⁺ cellproliferation.

H. Inhibition of Lymphocyte Proliferation in Response to AllogenicIrradiated Stimulator Cells

Monoclonal antibodies to ICAM-R were also tested for the ability toalter lymphocyte proliferation (as measured by ³ H-thymidineincorporation) in response to alloantigenic irradiated stimulator cells.Responder cells were prepared by obtaining PBMC from a normal donorusing Histopaque centrifugation as described above. To preparestimulator cells, PBMC from a second, unrelated donor were concurrentlyisolated and irradiated at 1500R by exposure to a gamma emitting cesiumsource. Two hundred thousand responder cells and 2×10⁵ irradiatedstimulator cells (suspended in culture medium) were then added to wellscontaining soluble or immobilized ICR-1.1, ICR-2.1, ICR-3.1, ICR-4.2,ICR-5.1, ICR-6.2, ICR-7.1, ICR-8.1, ICR-9.2, immobilized B-H9,immobilized p10.1, or soluble 515F (anti-rat CD18) antibody andincubated for 6 days at 37° C., 5% CO₂. Lymphocyte proliferation (³H-thymidine incorporation) was assessed in the last 18-24 hours ofculture.

Immobilized monoclonal antibodies ICR-1.1, 2.1, 6.2 and 8.1 consistentlyreduced proliferation in comparison to control antibodies. ICR-8.1 alsoinhibited alloantigen-stimulated proliferation when administered insoluble form.

I. Inhibition of IL-2 Production by T Lymphocytes

Human PBL were obtained by Ficoll-Hypaque centrifugation of wholeperipheral blood. Adherent cells were depleted by incubation on plasticand nonadherent cells were subjected to discontinuous centrifugation onPercoll gradients to further separate subsets of lymphocytes into mediumbuoyant density (fraction B) and high buoyant density (fraction C) cellpopulations. Prior to cell addition, wells were coated with monoclonalantibodies by addition of 0.1 ml each antibody at 5 ug/ml in PBS permicrotiter well. Following antibody addition, all wells were incubatedovernight at 4 degrees and each well was washed free of unbound antibodyby PBS rinsing prior to addition of cells. Each cell fraction was thenincubated for 18 hours on either ICR-1.1, ICR-4.2 or control antibodiesto human major Histocompatibility Complex Class I (MHC Class I). Forthese experiments 2×10⁵ cells per well were added in a volume of 0.2 mlRPMI-1640 containing 10% FCS to individual wells of a 96 well flatbottom microtiter plate (Costar, Cambridge, Mass.). After 18-20 hoursincubation at 37° C., the cells were collected from the microtiter wellsand washed twice in RPMI-1640 media containing 10% FCS and adjusted to1×10⁶ per ml in RPMI-1640 containing 10% FCS. Two×10⁵ prepulsed cellswere then added to wells previously coated with 0.1 ml anti-CD3 antibody(clone OKT3 at 5 ug/ml in PBS) and the cells were incubated for 20-24hours at 37 degrees. After incubation supernatants were obtained fromeach well and replicate supernatants were pooled, frozen at -80 degreesand assayed for IL-2 content by ELISA (Biosource).

Fraction C cells, composed largely of quiescent CD3 positive cells,produced ample IL-2 when prepulsed for 18 hours in wells containingeither no antibody or a variety of control antibodies to MHC Class I.Cells prepulsed on ICR-1.1, however, produced less than 50% of IL-2produced following prepulse on negative control antibodies while cellsprepulsed on ICR-4.2 exhibited no decreased IL-2 production. Thus, notall ICAM-R specific antibodies were efficacious in inhibiting IL-2release. Engagement of ICAM-R in an epitope specific manner is requiredfor this effect to be achieved. It is anticipated that ICAM-R specificantibodies whose binding sites on ICAM-R overlap significantly or areidentical to that bound by ICR-1.1 (e.g., ICR-8.1, see Example 14) wouldmanifest similar effects.

J. Restoration of Anti-CD3 Mediated Proliferation by Addition of IL-2

Human PBL were fractionated and incubated on immobilized monoclonalantibodies as described in Section I above. The cells were collected,washed and replated on anti-CD3 either in the presence or absence ofhuman rIL-2 at 30 U/ml. Addition of IL-2 completely restoredproliferative responses to anti-CD3 by resting lymphocytes prepulsed onICR-1.1, indicating that the inhibitory effect of ICR-1.1 was not due toirreversible toxicity to the cells.

K. Induction of IL-8 Release

Monocytes were isolated by elutriation from peripheral blood of normaldonors. Plastic wells were coated with ICR- 1.1, 2.1, 8.1 or albuminalone (10 μg/ml). After blocking free sites, the cells were placed intothe wells in medium. After 1 hour in culture, the cells that were in theICR Mab treated wells had flattened onto the substratum. Those cellsplated in wells treated with BSA plus or minus LPS were rounded and notspread. After 8 or 18 hours of incubation at 37° C., the medium wasremoved and assayed for IL-8 immunoreactivity. At the 8 hour time point,all medium tested from wells containing ICR monoclonal antibody showedenhanced levels of IL-8 (four times over albumin control). By 18 hours,IL-8 levels in media from ICR monoclonal antibody wells were muchelevated over control levels. ICR-1.1 induced levels increased tentimes, ICR-2.1 induced levels were four times and ICR-8.1 induced levelswere two times over the levels seen at the 8 hour time point. Similarexperiments with monocytic cell lines (U937 and HL60) to determine ifICR monoclonal antibody can induce IL-8 release were conducted. U937cells responded to each of the ICR monoclonal antibody treated wells byreleasing IL-8 into the conditioned medium. ICR-1.1 elicited the mostrobust response which was 3-fold greater than the release from ICR-4.2,6.2 or 8.1 treated wells, each of which showed levels twice that of theBSA control alone. HL60 cells did not respond to the ICR antibodytreated wells by releasing IL-8 into the medium. LPS did induce a markedrelease from HL60 cells. No detectable morphological changes weredetected with the U937 or HL60 cells.

Monocytes isolated and treated as described above were tested forrelease of MCP-1 at 8 and 18 hour time points. ICR-1.1, 2.1 and 8.1 eachinduced release of MCP-1 into the conditioned medium although thekinetics of release differed. MCP-1 release elicited by ICR-1.1 peakedat 8 hours. Release from Mab ICR-2.1 and ICR-8.1 was not detected until18 hours when it was 4-fold greater than the peak 1.1 induction level.

HL-60, U937, and the monocytic leukemic cell line THP-1 were alsoassayed for release of MCP-1 and MIP-1 alpha from conditioned mediaafter monoclonal antibody activation was performed as described forIL-8. After 18 hours, levels of MCP-1 and MIP-1 alpha were significantlyhigher than background in THP-1 containing wells, but not in U937 orHL-60 containing wells. The release of these cytokines was also assayedafter 4 and 8 hours. In most cases, the levels of cytokine in the mediarose between 4 and 8 hours. ICR 8.1 induced expression of both MIP-1alpha and MCP-1 was between 5 and 10 times background.

Monocytes from peripheral blood and HL-60, U937 and THP-1 cell lineswere assayed for TNF-alpha production after activation by anti-ICAM-Rmonoclonal antibodies in a manner similar to that described for IL-8activation. Four to 8 hours after treatment with monoclonal antibodyICR-S. 1 and 18 hours after treatment with monoclonal antibody ICR-8.1,monocytic release of TNF-alpha was elevated. HL60 cell release ofTNF-alpha 8 hours after treatment with ICR-8.1 was twice background.Both THP-1 and U937 cell release of TNF-alpha was elevated after 8 hoursof binding to ICR-8.1.

These results imply a potentially significant role of ICAM-R in thehuman disease atherosclerosis since engagement of ICAM-R in the presenceof a pro-atherosclerotic compound (e. g., oxidated phospholipid)promotes synthesis/secretion of MCP-1 which has recently been implicatedas a pro-atherosclerotic chemokine Edgington, BIO/TECHNOLOGY, 11:676-681(1993)!. MIP-1 alpha has been associated with early wound healing Faheyet al., Cytokine, 2:92 (1990)!; acute lung injury induced by immunecomplex administration in rodents Shanley, et al., J. Immunol.,154:4793-4802 (1995)!; allergic airway inflammation in rodents Lukacs etal., Eur. J. Immunol., 25: 245-251 (1995)! and during episodes ofmultiple sclerosis relapse Miyagishi et al., J. Neurol. Sci, 129:223-227(1995)!.

L. Upregulation of the Activation Antigens CD69 and CD25

Resting PBL isolated as described in Section I above were prepulsed onICR-1.1 or negative control antibodies, washed and incubated onimmobilized anti-CD3 for 24 hours. The cells were then collected,labeled with monoclonal antibodies to the lymphocyte activation antigensCD69, CD25 and CD80 and examined by flow cytometry. Cells prepulsed onICR-1.1 do not exhibit decreased ability to upregulate the activationantigens CD69 and CD25 in response to immobilized anti-CD3 compared tothe level of CD69 and CD25 expressed by cells prepulsed on controlantibodies.

M. Increased Tyrosine Phosphorylation in Human PBL

Human PBL was obtained by Ficoll-Hypaque centrifugation and wereincubated for 5 minutes with soluble ICR-4.2, OKT3 or anti-HLA-I (1×10⁷cells were incubated with each antibody at 30 ug/ml). The cells werethen washed and goat anti-mouse IgG (Cappell) was added to a finalconcentration of 100 ug/ml. After varying periods of time, the cellswere lysed in detergent and lysates were electrophoresed on a 10%acrylamide gel, transferred to blotting paper. Blots were then probedwith the anti-phosphotryosine antibody 4G10.

ICR-4.2 induced phosphorylation on tyrosine of numerous substratesrapidly after crosslinking compared to the negative control antibody toMHC Class I. Phosphorylation of substrates were also observed inresponse to the positive control (crosslinked OKT3 antibody).

N. Effect of ICAM-R Binding on Early Signaling Events in PBL

Given the ability of antibodies to domain 1 of ICAM-R to inhibitsubsequent T cell activation, intracellular T cell signaling pathwayswere examined in order to understand which signaling events are affectedby ICAM-R. More specifically, the ability of cross-linked T cell antigenreceptor to induce tyrosine phosphorylation was examined. T cells areknown to show a rapid induction of tyrosine phosphorylation in responseto antigen presentation or various cellular substrates. This inductionis known to be essential to subsequent proliferation and IL-2production.

Briefly, resting PBLs were isolated and treated with ICR-1.1 or isotypematched control antibody as described above in Section I. After thecells were removed from antibody, they were washed one time in PBS,resuspended in PBS to a concentration of 1×10⁶ cells/ml, and eithertreated or not treated with antisera to the T cell receptor (G19-4) for2 minutes at 37° C. The cells were then spun down and lysed in boiling2× SDS-sample buffer, boiled a further 5 minutes and resolved on 10%SDS-PAGE. Proteins were transferred to nitrocellulose and blotted withthe 4G10 anti-phosphotyrosine antibody as described. Regardless ofwhether the cells had been pretreated with ICR-1.1 or matched isotypeantibody, there was an induction of tyrosine phosphorylation in responseto G19-4 treatment. This suggests that, at least on a gross level,signaling through tyrosine kinases is normal in these cells. This isconsistent with the result that CD69 upregulation in these cells is alsonormal (Section L above), since CD69 expression requires PLC-gammastimulated PKC activity and PLC-gamma activation requires tyrosinephosphorylation. The notion that ICR-1.1 pretreatment inhibits asignaling event parallel on subsequent to PLC-gamma activation is alsosupported by the result that PMA/ionomycin treatment does not inducenormal T cell activation in the pretreated cells.

O. ICAM-R Engagement and Translocation of NFAT

Translocation of the transcription factor NFAT from the cytoplasm to thenucleus is essential for IL-2 gene transcription. The presence of NFATcomplexes in the nuclei of cells in response to ionomycin (a member of aclass of compounds which cause calcium transport across cell membranesand which can signal the Ca²⁺ dependent pathway associated with TCRstimulation) may be assayed as follows. Briefly, an oligonucleotidecorresponding to the IL-2 promoter of the human IL-2 gene isend-labelled with ³² p and purified. Proteins in the nuclear fraction ofionomycin-treated Jurkat cells are isolated and then incubated with theoligonucleotide. Resulting complexes are resolved on a non-denaturingPAGE gel. In this assay, a Jurkat cell line which produced IL-2 atnormal levels contained a transcription complex which formed in thepresence of ionomycin but, as expected, was not formed when cells werepretreated with cyclosporin A. A Jurkat cell line which did not produceIL-2 at normal levels failed to form this transcription complex inresponse to ionomycin.

This result is significant since the NFAT family of transcriptionfactors are thought to be the proximal targets for calcineurin.Calcineurin, in turn, is the intracellular target for cyclosporin A andFK506, two drugs which have been utilized to support tissuetransplantation. Since ICAM-R may engage the same pathway as calcineurinbut is expressed more selectively (e.g., on leukocytes) thancalcineurin, engagement of ICAM-R may have a more selective therapeuticeffect.

P. ICAM-R Specific Antibody Inhibits of Normal Alloantigen Presentationto CD4⁺ T Cells

The expression of ICAM-R in normal skin, psoriasis, atopic eczema andcutaneous T cell lymphoma was examined. Five μm cryostat sections ofskin were stained using monoclonal antibodies to ICAM-R (ICR-1.1 andICR-8.1) and a well characterized immunoperoxidase technique. In normalskin, ICAM-R was expressed by all cutaneous leucocytes but most strikingwas the strong expression of ICAM-R by Langerhans cells (Lcs) withinboth epidermis and dermis. This observation was confirmed bydouble-labeling with CD1a (a Langerhans cell marker) and negativestaining with an IgG₁ isotype control. In psoriasis, atopic eczema, andcutaneous T cell lymphoma ICAM-R was co-expressed in all CD 1a⁺ cells.

Blocking experiments were performed to determine whether the observedICAM-R expression on Lcs was functionally important in antigenpresentation. CD4⁺ T cells were prepared from peripheral blood and 10⁵CD4⁺ T cells were combined with 4×10⁴ epidermal cells harvested fromkeratome biopsies of normal skin of an individual allogenic to the Tcell donor. Proliferation was measured by ³ H thymidine uptake.Alloantigen presentation was unaffected by addition of 50 μg IgG₁isotype control. Addition of 50 μg anti-ICAM-R antibody ICR-8.1 to theco-culture resulted in a marked (47%) reduction in degree of Lcsalloantigen-driven proliferative response of the T cells. Inhibition was73% of that produced by addition of anti-LFA-1 (anti-CD11a) antibody.

Q. Potentiation of Basophil Mediator Release and Adhesion to EndothelialCells

Basophils participate in acute allergic reactions by virtue of theirability to release preformed and newly generated mediators as a resultof IgE-dependent stimulation. Because ICAM-R is expressed at significantlevels on basophils, crosslinking of ICAM-R with specific monoclonalantibodies was tested for its effect on production of the basophilmediators histamine, leukotriene (LTC₄) and IL-4 and on adhesion ofbasophils to endothelial cells.

Basophils were obtained at low purity by dextran sedimentation oferythrocytes in EDTA-anti-coagulated peripheral venous blood, atslightly enriched percentages from mononuclear cell layers of densitygradient Percoll centrifugation preparations, and at high purity (>70%)from leukophoresis packs following clutriation and density gradientcentrifugation. In the assays, polyclonal goat anti-human IgE and ICAM-Rmonoclonals antibodies ICR-1.1, ICR-2.1, ICR-4.2 and ICR-8.1 were usedalone or in combination. The human IgE was used to induce histaminerelease which was quantitated using an automated fluorometric assayafter a 45 minute incubation with secretagogue. LTC₄ production wasmeasured by radioimmunoassay as previously described in Schleimer etal., J. Immunol., 143:1310-1317 (1989), while IL-4 in cell supernatantswas assayed using the IL-4 Quantikine ELISA kit (R&D Systems,Minneapolis, Minn.). Basophil adhesion to unstimulated or IL-1 treatedmonolayers of human umbilical vein endothelial cells was examined aspreviously described in Bochner et al., J. Clin. Invest., 81: 1355-1360(1988) and Bochner et al., J. Immunol., 142: 3180-3186 (1989), in thepresence or absence of ICR-2.1 monoclonal antibody. For basophilmediator release assays, ICR antibody was added simultaneously withanti-IgE or cells were pre-incubated for up to 30 minutes at 37° C. withICR antibody prior to addition of secretagogue.

In initial studies it was determined that the simultaneous addition ofICR-2.1 monoclonal antibody with anti-IgE had the greatest potentiatingeffect on anti-IgE induced histamine release. The ability to potentiatehistamine release declined the longer the incubation period was extended(studied up to 30 min.). When ICR-2.1 antibody was added simultaneouslywith a concentration of anti-IgE that induced approximately half maximalhistamine release (i.e., 0.01 μg/ml), the monoclonal antibody inducedconcentration-dependent enhancement of anti-IgE-induced histaminerelease from human basophils. ICR-2.1 antibody alone did not inducehistamine release. Similar results were obtained with ICR-1.1 monoclonalantibody, while neither ICR-8.1 nor the domain 2-specific monoclonalantibody ICR-4.2 had any significant potentiating activity.

ICR-2.1 monoclonal antibody also enhanced anti-IgE-induced basophil LTC₄production. As was seen with histamine release, ICR-2.1 antibodytreatment alone (in the absence of anti-IgE) failed to induce basophilLTC₄ production.

When basophil production of IL-4 was examined, somewhat differentresults were obtained. In the presence of anti-IgE alone, basophilsreleased 31 pg of IL-4 per million basophils. In the presence of ICR-2.1monoclonal antibody alone (6.6 μg/ml) and in the absence of anti-IgE,basophils released 80 pg of IL-4 per million basophils. Co-incubation ofanti-IgE and ICR-2.1 resulted in slightly higher release of IL-4 (96pg/million basophils).

When the ability of ICR-2.1 antibody to alter basophil adhesion tounstimulated or IL-1 (5 ng/ml, 4 hours, 37°0 C.) treated umbilical vienendothelial cell monolayers was examined, spontaneous adhesion ofbasophils to unstimulated endothelial cells was 12% and increased to 15%and 21% in the presence 2.2 and 6.6 μg/ml of ICR-2.1 antibody,respectively. Adhesion to IL-1 stimulated endothelium in the absence ofmonoclonal was 51% and increased to 67% and 83% in the presence of 2.2and 6.6 μg/ml of ICR-2 antibody, respectively.

The foregoing experiments illustrate that crosslinking of ICAM-R isassociated with a potentiation of anti-IgE-induced histamine relase andLTC₄ production. Additionally, anti-ICAM-R treatment alone inducessignificant IL-4 production from human basophils and may potentiateanti-IgE-indcuded IL-4 production from these cells. Finally, treatmentof basophils with ICR-2.1 antibody enhanced adhesion of basophils tounstimulated and IL-1 activated endothelial cells. Thus, crosslinking ofICAM-R on basophils potentiates a number of biological activities,including mediator release and adhesion.

EXAMPLE 21

Table 11 below is a summary of certain characteristics of ICAM-Rspecific monoclonal antibodies of the invention which have beenspecifically described in the foregoing examples. In Table 11, theabbreviation "NC" stands for "not conclusive" and the abbreviation "ND"stands for "not determined." The antibodies marked with an asterisk inTable 11 enhanced activation at low concentrations.

                                      TABLE 11                                    __________________________________________________________________________    Produced            Residues      Blockade of Adhesion                                                                    Blockade of                       by             Reactive                                                                           Critical/Important to                                                                       of JY Cells to                                                                          Lymphocyte Adhesion               Antibody                                                                           Hybridoma                                                                           Isotype                                                                           Domain                                                                             Binding       Soluble ICAM-R                                                                          SEA Alloantigen                   __________________________________________________________________________    ICR-1.1                                                                            26E3D IgG.sub.2a                                                                        1    F21V, E32K, E37T,                                                                           YES       YES YES                                               K33I, W51A, Y70                                           ICR-2.1                                                                            26H11C                                                                              IgG.sub.1                                                                         1    F21V, E32K, K33I,                                                                           NO        YES YES                                               W51A, Y70                                                 ICR-3.1                                                                            26I8F IgG.sub.1                                                                         1    F21V, E32K, E37T, Y70                                                                       YES       NO  NC                            ICR-4.2                                                                            26I10E                                                                              IgG.sub.1                                                                         2    F21V          NO        NO  NC                            ICR-5.1                                                                            42C5H IgG.sub.2a                                                                        1    F21V, E37T, W51A, Q75I                                                                      YES       YES NC                            ICR-6.2                                                                            42D9B IgG.sub.1                                                                         2    F21V, W51A    NO        YES YES                           ICR-7.1                                                                            43H7C IgG.sub.1                                                                         1    F21V, E37T, W51A,                                                                           NO        NO  NO                                                Y70, Q75I, E32K, K42E, L44V                               ICR-8.1                                                                            46D7E IgG.sub.1                                                                         1    F21V, E32K, W51A                                                                            YES       YES YES                           ICR-9.2                                                                            46I12H                                                                              IgG.sub.2a                                                                        2    F21V          NO        NO  NO                            ICR-12.1                                                                           63E11D                                                                              IgG.sub.1                                                                         1    ND            YES       YES*                                                                              ND                            ICR-13.1                                                                           63G4D IgG.sub.1                                                                         1    ND            YES       NO* ND                            ICR-14.1                                                                           63H4C IgG.sub.1                                                                         1    ND            YES       NO* ND                            ICR-15.1                                                                           63H6H IgG.sub.1                                                                         1    ND            YES       NO* ND                            ICR-16.1                                                                           63I1C IgG.sub.1                                                                         1    ND            YES       YES*                                                                              ND                            ICR-17.1                                                                           6316G IgG.sub.1                                                                         1    ND            YES       YES ND                            ICR-19.3                                                                           81K2F IgG.sub.1                                                                         3    ND            NO        NO  ND                            __________________________________________________________________________

EXAMPLE 22

One inference from the aforementioned examples that antibodies specificfor ICAM-R modulate the response of lymphocytes to a variety of stimuli(e.g., SEA and allogeneic cells) is that engagement of ICAM-R by eitherits natural counter-receptors or by antibodies of the inventiontransduces a signal to the ICAM-R expressing cell. ICAM-R specificsignaling events are likely to involve the interaction of thecytoplasmic domain of ICAM-R with cellular enzymatic components (e.g.,kinases, phosphatases) of one or more second messenger pathways and/orwith cytoskeletal components in a pattern unique to ICAM-R.

A. Phosphorylation of ICAM-R

Preliminary experiments are consistent with this concept and with theidea that ICAM-R is distinct from ICAM-1 in its linkages with secondmessenger systems. Extracts from unstimulated Raji cells were prepared,fractionated and assayed for kinase activity as follows. Seven×10⁷ cellswere washed once in PBS and lysed in buffer containing 20 mM Tris pH7.5, 0.5 mM EDTA, 1% Triton X-100 (Pierce), 10 ug/ml pepstatin andleupeptin (Boehringer), 2 mM PMSF for 1 hour on ice. Lysates werepelleted in a refrigerated microfuge at 14,000 rpm for 15 minutes andthe resulting supernatant was applied to a DEAE sephacel column(Pharmacia) equilibrated in 20 mM Tris pH 7.5, 0.5 mM EDTA (Buffer A).The column was run at a rate of 0.25 ml/minute and developed with agradient of 0 to 0.35M NaCl in buffer A over 60 minutes. In theseinitial experiments, only those fractions enriched in protein kinase C(PKC) activity (as determined using an Amersham assay kit and followingmanufacturers instructions) were examined. Fractions enriched in PKCactivity were pooled and used as a source of kinase(s) to test fordifferential phosphorylation of synthetic peptides of the completecytoplasmic domains of ICAM-1, ICAM-2 and ICAM-R (amino acids 481 to 518of SEQ ID NO: 1). Assays were performed according to manufacture'sinstructions with peptides at 75 uM final concentration. Ten ul of thereaction mixture was boiled in 30 ul Laemmli sample buffer and resolvedon a 12.5% SDS-PAGE gel. Following a 1.5 hour exposure of the gel onX-ray film phosphorylation of ICAM-R and ICAM-2 but not ICAM-1 wasdetected. Whether the phosphorylation was due to PKC or anotherco-fractionated kinase was not determined.

Further assays involved reacting fractions derived either from a columnchromatography step or from solubilized cell fractions in the presenceof Ca⁺⁺, Mg⁺⁺, cAMP, phosphatidylserine, cytoplasmic tail peptide and ³²P!ATP. Phosphorylation of specific peptides was assessed followingresolution by gel electrophoresis. Jurkat cells were separated intosubcellular fractions and each fraction was assayed for kinase activityon the cytoplasmic tail peptides. In these experiments, phosphorylationof ICAM-1 and ICAM-R was detected. However, kinases which phosphorylatedICAM-1 associated with cell membrane fractions, whereas kinases whichphosphorylated ICAM-R were primarily cytosolic although also present inmembranes. Additional support for different kinases acting on these twoICAM's comes from preliminary purification studies of these kinases.Jurkat cytosol fractionated on a MonoQ column (Pharmacia) equilibratedin 50 mM Tris pH 8, 5 mM EDTA and developed with a gradient to 0.6M NaClover 30 minutes gives a very broad activity profile for kinases actingon ICAM-R. Only a subset of these fractions also have activity towardsICAM-1. This provides additional evidence that cellular kinases existwhich differentially phosphorylate ICAM-R but not ICAM-1. Twodimensional phosphoamino acid analysis on these phosphorylated peptidesshows only serine phosphorylation on ICAM-R and threoninephosphorylation on ICAM-1.

The ability of the protein tyrosine kinases p56^(lck) (UBI, Lake Placid,N.Y.), and p59^(fyn) (UBI, Lake Placid, N.Y.) to phosphorylate acytoplasmic tail peptide (amino acids 482 to 518) of ICAM-R was measuredin vitro.

The assay was performed in triplicate as follows. Five μl assay bufferstock solution (250 mM Tris pH 6.8, 125 mM MgCl₂, 25 mM MnCl₂, 0.25 mMNa₃ VO₄), 5 μl cdc 2 (UBI, positive control) or ICAM-R cytoplasmic tailpeptide or scrambled ICAM-R cytoplasmic tail peptide or H₂ O, 10 μl (1u) p56^(lck) or p59^(fyn), and 5 μlATP stock solution (0.25 μCi γ³² PATPin 500 μM cold ATP) were mixed in a microfuge tube and incubated 30minutes at 30° C. Ten μl 50% acetic acid were then added. Samples of 25μl were spotted on P81 phosphocellulose paper and washed four times with150 mM phosphoric acid. The papers were then dried and Cerenkov countedfor one minute.

p59^(fyn) phosphorylated the cytoplasmic tail of ICAM-R (but not thescrambled cytoplasmic tail peptide) and did so approximately 1.8 timesbetter than it phosphorylated an equimolar amount of cdc 2 peptide.p56^(lck) failed to phosphorylate any of the substrates at aconcentration of 1 u (unit), but was able to phosphorylate ICAM-Rcytoplasmic tail peptide and scrambled peptide at concentrations of 10u.

Jurkat cells were labeled in vivo with ³² P orthophosphate for threehours with and without the mitogen PHA (phytohemagglutinin). The cellswere lysed and ICAM-R was immunoprecipitated from the lysates. Bothsamples showed incorporation of radioactive phosphate, however, thesample incubated with PHA showed at least a five fold increase inradioactive phosphate incorporation. Phosphoaminoacid analysis indicatedthat both samples contained only phosphoserine as the phosphoacceptorsite.

B. Association with Cytoskeletal Components

Preliminary experiments also indicate that the cytoplasmic domain ofICAM-R differentially associates with cytoskeletal components. Bindingof the non-competing monoclonal antibodies ICR-1.1 and ICR-4.2 to ICAM-Rwas examined to assess the potential influence of each antibody on theassociation of lymphocyte ICAM-R with the cytoskeleton. The antibodiesmay mimic distinct natural ICAM-R ligands which employ ICAM-R as a cellsurface receptor through which regulated cellular responses may beelicited.

Other investigators have previously observed that numerous human Tlymphocyte surface antigens which occur as cell surface transmembraneglycoproteins can be induced to associate with the cytoskeleton if cellsurface-bound antibody specific for these antigens is crosslinked withsecondary antibodies Geppert et al., J. Immunol., 146: 3298 (1990)!.Many of these cell surface molecules are defined components oflymphocyte adhesion and/or activation pathways. The phenomenon ofinducible association with the cytoskeleton is operationally defined asthe resistance of cell-surface immune complexes to detergent extractionunder defined conditions. Inducible detergent resistance does notrequire metabolic energy and can be observed in cells maintained at0°-4° C. throughout the experiment.

Experiments were conducted using freshly prepared human PBL or the humanT lymphoblastoid cell line CEM-CCRF (ATCC CCL119). Briefly, freshlydrawn human blood from healthy volunteer donors was diluted 1:1 with PBSand layered onto Sigma HistoPaque density separation medium. Thegradients were centrifuged for 30 minutes at 1500 rpm (600×g) and themononuclear cell fraction at the interphase was collected and washedthree times with PBS. The cell pellet was resuspended in completeRPMI-1640 medium (Gibco, supplemented with L-glutamine,penicillin/streptomycin, sodium pyruvate, 2-mercaptoethanol, and 10%FBS) and plated onto tissue culture-treated petri dishes for adherentcell depletion. Plates were incubated 1-2 hours at 37° C., 5% CO₂ afterwhich nonadherent PBL were harvested and washed twice with ice-cold PBS.Conjugation of monoclonal antibodies to fluorescein using fluoresceinisothiocyanate (FITC) was performed according to published proceduressee, e.g., Goding, J. Immunol. Meth., 13: 215 (1976)! and, in brief,involves incubation of purified antibody with an excess of FITC (Sigma)in 0.1M bicarbonate buffer pH 8.1 for 90 minutes at 37° C. followed byexhaustive dialysis against PBS to remove unreacted FITC.

PBL or washed CEM cell suspensions (1×10⁶ cells) were dispensed intoFalcon 12×75 mm tubes in ice-cold PBS-5% FBS, pelleted, and resuspendedin 50 μl of FITC-conjugated anti-ICAM-R monoclonal antibody 26E3D-1 or26I10E-2 adjusted to saturating concentration in the same buffer.Antibody binding was permitted to proceed for 30 minutes on ice,afterwhich unbound antibody was removed by pelleting cells which hadfirst been resuspended in 1 ml of PBS-5% FBS through an underlaidcushion (0.7 ml) of neat (undiluted) FBS.

For groups stained with FITC-conjugated monoclonal antibody only, the 1ml suspension was divided into two equal parts, each of which wasseparately underlaid with FBS, centrifuged, and the supernatant removedby aspiration. Cell pellets were then resuspended in 200 ul of controlbuffer (13 mM Tris pH 8.0, 150 mM NaCl, 2 mM MgCl₂, 2 mM EGTA, 2% FBS,2.5 ug/ml aprotinin, 1 mM PMSF, 10 mM iodoacetamide) or detergent buffer0.5% NP-40 (v/v) (US Biochemical, Cleveland, Ohio) in control buffer!and held for 20 minutes at room temperature, or overnight at 4° C.,prior to FACS analysis. For groups in which cell surface-boundmonoclonal antibody was crosslinked with secondary antibodies, followingthe first antibody staining step, washed cell pellets were resuspendedin 50 ul of FITC-goat anti-mouse IgG (Sigma) diluted 1:100 in PBS-5% FCSand incubated for 30 minutes on ice. The cells were then resuspended,divided into two tubes as described above, pelleted, and buffer-treatedin the presence or absence of detergent. FACS analysis was thenperformed on the cells.

Results (see FIG. 15) obtained for CEM cells were similar to those seenwith PBL. ICAM-R association with the cytoskeleton as assessed by thedetergent resistance assay was negligible when FITC-conjugated ICR-4.2or ICR-1.1 antibodies alone were permitted to bind to cell surfaceICAM-R. However, when cell surface-bound ICR-4.2 antibody wascrosslinked with secondary antibodies, a modest increase in detergentresistance was detected. If secondary antibodies were used to crosslinkcell surface-bound ICR-1.1, which recognizes a distinct ICAM-R epitopefrom that seen by ICR-4.2, a much greater (approximately 2-fold in PBLand 2-3 fold in CEM) increase in detergent resistance was reproduciblyobserved. Interaction of ICAM-R ligands with different structuralregions of ICAM-R thus appears to differentially influence associationof ICAM-R with the cytoskeleton.

In addition, initial experiments have shown that by enriching the poolof phosphotyrosyl proteins in Jurkat 77 cells with tyrosine phosphataseinhibitors or by T-cell receptor activation, it is possible to observephosphotyrosyl proteins co-immunoprecipitating with ICAM-R. By Westernblotting with an antibody to phosphotyrosine, one band in particularmigrated in the range of the tryosine kinase ZAP-70 (zeta chainassociated protein) Chan et al., Proc. Natl. Acad. Sci. USA, 88:9166-9170 (1991)!. The zeta chain is associated with a tyrosine kinaseand upon T cell antigen receptor stimulation associates with Zap70, a 70kDa tyrosine phosphoprotein. In an attempt to identify thisphosphotyrosyl protein, Jurkat cells were stimulated with PHA(phytohemagglutinin) or with T-cell receptor cross linking using theantibody OKT3 and lysed at various time points. ICAM-R wasimmunoprecipitated from these lysates, run on SDS-PAGE, tranferred to aPVDF membrane, and probed with an antibody to ZAP-70. As early as fiveminutes post stimulation with PHA and ten minutes with OKT3crosslinking, a band immunoreactive with the ZAP-70 antibody could beseen co-immunoprecipitating with ICAM-R in a transient fashion.

EXAMPLE 23

Characterization of ICAM-R interaction with specific cytoplasmicproteins was conducted.

A. Dihybrid Screen

The two-hybrid system developed in yeast Chien et al., Proc. Natl. Acad.Sci. USA, 88: 9578-9582 (1991)! was used to screen for products of ahuman lymphocyte cDNA library capable of interacting with thecarboxy-terminal cytoplasmic tail of ICAM-R. This yeast dihybrid screenis based on functional in vivo reconstitution of the GAL4 transcriptionfactor. The separable DNA-binding and transcription-activating domainsof GAL4 were engineered into distinct plasmids as portions of novelfusion proteins. Under defined conditions GAL4 activity is measureableby assay of the beta-galactosidase reporter gene.

One plasmid, the "bait" vector (pAS1), contained sequences encoding theGAL4 DNA-binding domain amino acids 10-147, Keegan et al., Science231:699-704 (1986)!, a -trp requirement, the HA epitope tag, and apolylinker region into which the ICAM-R cytoplasmic domain sequence wasligated at the BamHI site. The ICAM-R cytoplasmic domain was amplifiedby PCR from pVZ-147 ICAM-R DNA (Example 4) using the oligonucleotideprimers:

DH3 (SEQ ID NO: 111)

CAGTGGGATCCTGTTAATGTACGTCTTCAGGG and

DH4 (SEQ ID NO: 112)

TGGGAGTTTGAAGGCTTT.

and then inserted at the BamHI site. The resulting construct, termedplasmid 9.4, was sequenced to confirm orientation and rule out PCRerrors. Yeast strain Y190 (genotype MATα gal4 gal80 his3 trp1-901ade2-101 ura3-52 leu-3,-112 +URA3::GAL→lacZ, LYS2::GAL→HIS3 cyh^(r)) wastransformed with plasmid 9.4 by standard methods and grown in selective(-trp) media to mid-log phase. Cells were lysed with glass beads inlysis buffer and 50 ug of protein was loaded onto a 10% polyacrylamidegel which was electrophoresed and blot-transferred to a PVDF (Millipore)membrane by standard procedures. Control lanes of the gel containedlysate material from pAS1-transformed Y190. Blots were developed usinganti-HA monoclonal antibody 12Ca5 (BAbCo, Berkeley, Calif.), rabbitanti-mouse IgG, and ¹²⁵ I-labeled protein A to confirm that the chimericfusion protein (ICAM-R cytoplasmic tail/HA/GAL4 DNA-binding domain) wasexpressed at readily detectable levels.

The second expression plasmid, or "prey" vector (pACT), consists ofsequences encoding the GAL4 activation domain II amino acids 768-881, Maet al., Cell, 48:847-853 (1987)! fused to a human B cell cDNA libraryinserted at the Xho site of the vector, and a-leu selection requirement.

The 9.4-transformed Y190 cells were transformed by standard methods withpACT library DNA and grown under selective conditions(-leu/-trp/-his/3-aminotriazole). Only cells in which an interactionoccurred between the ICAM-R cytoplasmic tail domain of the 9.4 chimericprotein product and an unknown (B cell cDNA library-derived) proteinsequence fused to pACT GAL4 domains survived. This interaction wasrequired to reconstitute GAL4 activity.

Fifty colonies grew using this selection method and were tested forbeta-galactosidase activity. Specificity of the ICAM-R cytoplasmic tailinteraction with pACT fusion proteins was verified by inability of thelatter to complement recombinant pAS1 vector expressing distinct "bait"proteins p53, ICAM-1, ICAM-2, a kinase (surose non-fermemtor 1, snfl),and casein kinase inhibitor (CKIΔ or CKIa)! using the dihybrid selectionconditions described above. Sequence analysis of B cell cDNA-derived,pACT inserts obtained by this method revealed twenty novel sequences andthirty sequences encoding known proteins out of the fifty inserts.

Two of the known proteins, alpha-tubulin and protein kinase C inhibitorprotein (PKCIP), were further investigated for their ability to interactwith ICAM-R cytoplasmic tail. Alpha-tubulin, along with beta-tubulin, isa principal component of cytoplasmic microtubules, one major class ofpolymeric cytoskeletal proteins. The PKCIP which interacted with ICAM-Rcytoplasmic has a sequence identical to that of human HS1-beta (GeneProAccession No. gp x57346), a known phospholipase and member of the highlyconserved 14.3.3 family of PKC regulatory proteins.

Mutagenesis was employed to map the ICAM-R cytoplasmic tail sitesresponsible for interaction with the alpha-tubulin and PKCIP pACTplasmid products. Four ICAM-R cytoplasmic tail mutant sequences wereproduced in pAS1 by in vitro mutagenesis of plasmid 9.4 using thefollowing mutagenic oligonucleotides wherein, for example, the E495Doligonucleotide introduces an aspartic acid at position 495 which is aglutamic acid in wild-type ICAM-R:

E495D (SEQ ID NO: 113)

TACATGTTAGGGAGGACAGCACCTAT,

E494D (SEQ ID NO: 114)

TACCATGTTAGGGACGAGAGCACCTAT,

E495A (SEQ ID NO: 115)

TACCATGTTAGGGAGGCCAGCACCTAT,

E494 (SEQ ID NO: 116)

TACCATGTTAGGGCCGAGAGCACCTAT.

The resulting plasmids were transformed into Y190 cells, andcotransformed with either the alpha-tubulin or the PKCIP pACT plasmid.All contransformants grew in the selective medium described above andtested positive for beta-galactosidase activity, indicating that thesemutations did not disrupt interactions between the GAL4 fusion proteinsencoded by each plasmid.

Additional mutant 9.4 plasmids shown in Table 12 below were constructedby similar methods and were cotransformed into Y190 cells with thepositive bait vectors tubulin (GenePro gp K00558), EF-8 (GenePro gpM27364), EF-11 (GenePro gp 29548), HS1-beta (SWISS-PROT sp 27348),HS1-theta (SWISS-PROT sp 27348), actin (PIR-Protein pir 505430),triose-6-isomerase (SWISS-PROT sp P00939), and proteosome (SWISS-PROT spP25786)! that had been identified in the original screening of the cDNAlibrary. Transformants were tested for β galactosidase activity in orderto map residues in the ICAM-R cytoplasmic domain contributing to eachinteraction. The results are illustrated in Table 12 wherein "blue"indicates no effect, "white" indicates a complete disruption and "Lbal"indicates a minimal disruption in an interaction and mutations R482-R493and R482-Q506 represent the amino acid residues remaining after grossdeletions of other amino acids of the ICAM-R cytoplasmic tail.

                                      TABLE 12                                    __________________________________________________________________________                                                             PACT                 MUTATION                                                                            TUBULIN                                                                              EF-8   EF11                                                                              HS1-beta                                                                           HS1-theta                                                                          ACTIN                                                                             TRIOSE-6-ISOMERASE                                                                       PROTEOSOME                                                                            CONTROL              __________________________________________________________________________    MQ504/AA                                                                            WHITE  ND     BLUE                                                                              BLUE BLUE WHITE                                                                             BLUE       BLUE    WHITE                QP505/AA                                                                            WHITE  BLUE   BLUE                                                                              WHITE                                                                              WHITE                                                                              BLUE                                                                              WHITE      BLUE    WHITE                Q505/A                                                                              WHITE  WHITE  BLUE                                                                              BLUE BLUE WHITE                                                                             BLUE       BLUE    WHITE                Y498/A                                                                              WHITE/LBal                                                                           WHITE/BLal                                                                           BLUE                                                                              BLUE BLUE BLUE                                                                              BLUE       BLUE    WHITE                T502/A                                                                              WHITE/LBal                                                                           WHITE  BLUE                                                                              BLUE BLUE WHITE                                                                             WHITE      WHITE   WHITE                M504/A                                                                              WHITE  BLUE   BLUE                                                                              BLUE BLUE WHITE                                                                             BLUE       BLUE    WHITE                P506/A                                                                              BLUE   BLUE   BLUE                                                                              BLUE BLUE BLUE                                                                              WHITE      BLUE    WHITE                M504/G                                                                              BLUE   BLUE   BLUE                                                                              BLUE BLUE BLUE                                                                              BLUE       BLUE    WHITE                L501/A                                                                              BLUE   BLUE   BLUE                                                                              BLUE BLUE BLUE                                                                              BLUE       BLUE    WHITE                Y490/A                                                                              WHITE  BLUE   WHITE                                                                             BLUE BLUE WHITE                                                                             BLUE       BLUE    WHITE                R482-R493                                                                           WHITE  WHITE  WHITE                                                                             WHITE                                                                              WHITE                                                                              WHITE                                                                             WHITE      WHITE   WHITE                R482-Q506                                                                           BLUE   BLUE   BLUE                                                                              BLUE BLUE BLUE                                                                              BLUE       BLUE    WHITE                Y490/F                                                                              WHITE  BLUE   BLUE                                                                              BLUE BLUE WHITE                                                                             BLUE       BLUE    WHITE                Y498/F                                                                              WHITE  BLUE   BLUE                                                                              BLUE BLUE ND  BLUE       BLUE    WHITE                __________________________________________________________________________

For single and double point mutations, the interaction of the 14.3.3proteins HS1-beta and HS1-theta, are only disrupted (i.e., white) by thechange in mutant QP505/506 while the interaction with tubulin wasdisrupted by a variety of changes to the cytoplasmic tail. MutationsR482-R493 and R482-Q506 are gross deletions of the cytoplasmic tail. Thedeletion of amino acids 506 through 518 appears to not disrupt theinteractions tested, while deletion of amino acids 493 to 518 appears todisrupt all interactions. Two additional bait vectors respectivelyencoding the ICAM-1 and ICAM-2 cytoplasmic tails were constructed bysimilar methods to that described for ICAM-R to further test thespecificity of the alpha-tubulin and PKCIP interactions with the ICAM-RC-tail. When Y190 transformants expressing either the ICAM-1 or ICAM-2cytoplasmic tail fusion proteins were cotransformed with thealpha-tubulin or PKCIP pACT plasmids described above, there was noevidence for interactions among fusion proteins, indicating specificityof the original ICAM-R interactive proteins.

B. ICAM-R C-tail affinity chromatography

Additional direct evidence for binding interactions between ICAM-Rcytoplasmic tail and alpha-tubulin was obtained by a variety of methods.An ICAM-R cytoplasmic tail (C-tail) peptide corresponding to ICAM-Ramino acids 482-518 of SEQ ID NO: 1 was synthesized by MacromolecularResources, (Colorado State Univ., Boulder, Colo.) . The peptide wasimmobilized (19 mg/ml suspension) on agarose beads (AffiGel 10, BioRad)according to manufacturer's instructions. Detergent lysate (20 ml, lysisbuffer: 50 mM octyl glucoside, 50 mM Tris pH 7.5, 0.15M NaCl, 1 mMMgCl₂, 1 mM CaCl₂ and a protease inhibitor cocktail) of freshly isolatedhuman tonsils (12 gm) was applied first to an ethanolamine-blockedAffiGel 10 precolumn and then to the ICAM-R C-tail beads (0.5 ml) for 3hours at 4° C. Beads were batch-washed with over 50 bed volumes of lysisbuffer and packed into a glass column which was then eluted with 1 ml ofsoluble ICAM-R C-tail peptide (2.5 mg/ml lysis buffer). Aliquots of theeluate were analyzed by standard western immunoblot analysis followingSDS-PAGE using monoclonal antibodies specific for alpha-or beta tubulin(Sigma). Only alpha-tubulin was detected in the C-tail peptide eluateusing the ECL detection system (Amersham). Silver-staining of the eluatefraction proteins resolved in SDS-PAGE revealed that additional proteinswere present.

In another series of experiments, Jijoye cells were lysed at 30×10⁶ /mlin a buffer (HL) containing 1% Triton X-100, 10 mM HEPES, pH7.5, 42 mMKCl, 5 mM MgCl₂, 20 μM NaF, 1 mM Na₃ Vo₄ and a protease inhibitorcocktail for 15 minutes at 4° C. The lysates were then centrifuged at45K rpm for 30 minutes in a TL100 BECKMAN table top ultracentrifuge. Thehigh speed supernatant was then rotated for 2 hours at 4° C. with 100 μlof ethanolamine-blocked AFFIGEL 10 beads equilibrated in HL buffer. Thebeads were then spun onto SPIN-X 0.22 μm centrifuge filter units(Costar; 6K rpm for 2 minutes). The flow through was split equally andeach half rotated for 3 hours at 4° C. with 100 μl of either C-tailbeads (13 mg/ml) or scrambled C-tail beads (12.2 mg/ml). The beads werethen collected on SPINEX filters and sequentially eluted with 0.6 mg/mlof soluble scrambled C-tail peptide, 0.6 mg/ml and 1.2 mg/ml of solubleC-tail peptide. Beads were finally eluted with 200 μl of two timesconcentrated SDS-sample prep buffer. Aliquots of each eluate wereanalysed by standard Western immunoblot analysis following SDS-PAGEusing a monoclonal antibody specific to α-tubulin (Sigma) α-tubulin wasbound only to the C-tail beads and could be eluted only with solubleC-tail peptide.

C. ICAM-R C-tail Affinity Precipitation of Purified Cytoskeletal Protein

Purified alpha-beta dimer tubulin, alpha-actinin, and vinculin wereradioiodinated to specific activities of 1.9×10⁸ CPM/nMol, 0.3×10⁶CPM/nMol, and 4×10⁷ CPM/nMol, respectively, with ¹²⁵ 1 radionuclide(Dupont NEN). Affinity interaction of each of these radiolabeledproteins with ICAM-R C-tail beads was assayed using conditions (0.5%Tween-20, 50 mM Tris pH 7.5, 0.15M NaCl, 1 mM MgCl₂, 1 mM CaCl₂)previously employed to demonstrate specific ICAM-1 cytoplasmic tailassociation with alpha-actinin Carpen et al., J. Cell BioL, 118:1223-1234 (1992)!. Briefly, 20 μl of C-tail beads (19 mg peptide/mlresin) were incubated for 4 hours at 4° C. with 60×10³ cpm of ¹²⁵I-protein in the above-described Tween-20 buffer. After incubation, thebeads were spun onto 0.45 μm filtration units (Milliport; 6K rpm for 2minutes), the flow through collected and the beads washed three timeswith 50 mM Tris pH 7.5, 1 mM MgCl₂, 1 mM CaCl₂, 0.15M NaCl and 1% TritonX-100. Finally, the beads were boiled in 60 μl of 2 times concentratedSDS-sample prep buffer. Five μl aliquots of column flow through and SDSelutions were counted in a Beckman gamma counter.

When input CPM of radiolabeled proteins were standardized, only tubulinexhibited specific binding to ICAM-R C-tail.

EXAMPLE 24

ICAM-R cytoplasmic domain over-expression studies were performed toelucidate the functional consequences of ICAM-R C-tail molecularinteractions occurring inside cells which undergo phenomena such as Tcell receptor activation and cell-cell adhesion. The effect ofoverexpression of ICAM-R R C-tail within a cell type expressingendogenous levels of wild type ICAM-R on its surface was tested. Incontrast to effects on proliferation and IL-2 secretions observed whenresting PBLs were pretreated with ICR-1.1 (Example 21) concomitanttreatment of a lymphoblastoid cell line with ICR-1.1 and anti-CD3antibody resulted in co-stimulation of IL-2 production. Additionalstudies (see Example 21) utilizing the same combination of monoclonalantibodies have demonstrated a co-stimulatory role (probably via asecond messenger cascade) for ICAM-R in normal human PBL. The specificinteraction of the ICAM-R C-tail with alpha tubulin (see Example 23) asdetermined by biochemical criteria, may serve to anchor the membranephase with the cytoskeleton or to co-localize signaling molecules insidethe cell with those transmembrane proteins involved in cell-cellinteractions, as is known to occur in focal adhesive interactions withthe extra cellular matrix. Thus, ICAM-R appears to mediate secondmessenger signaling, probably via C-tail interactions withkinases/phosphatases and/or the cytoskeleton.

A. Cytoplasmic Domain Constructs

The following DNA constructs were made using the PCR-based method calledsynthesis by overlap extension Horton et al., Gene, 77: 61-68 (1989)!:haWT₄₅₃₋₅₁₈, haWT₂₈₆₋₅₁₈, and haCTΔ₂₈₆₋₄₈₄. The following nomenclaturehas been used for the constructs. "Ha" denotes an epitope tag sequencefrom the influenza hemagglutinin protein, which has been repeated threetimes in tandem to increase binding affinity with a commerciallyavailable monoclonal (12CA5, Boehringer Mannheim, Indianapolis, Ind.)antibody. "WT" refers to the native ICAM-R amino acid sequences. "CTΔ"refers to a deletion of the cytoplasmic tail. The numbers in subscriptdenote the starting and ending amino acids from ICAM-R included in therespective protein. Restriction enzyme cloning sites (HindIII and NotI)were engineered into the DNA constructs for subcloning into theexpression vector pRC/CMV (Invitrogen Corp., San Diego, Calif.) undercontrol of the cytomegalovirus immediate early enhancer/promoter. ThepRC/CMV plasmid also contains the bacterial neomycin resistance genefrom Tn5, thus allowing selection and maintenance of stable DNAintegration into the cellular genome. The DNA constructs for haWT₂₈₆₋₅₁₈and haCTΔ₂₈₆₋₄₈₄ plus other C-tail deletions were also subcloned into asecond expression vector pMHneo Hahn et al., Gene 127: 267-268 (1993)!which drives expression from the Friend spleen focus-forming virus longterminal repeat. This vector provides for high levels of proteinexpression in the Jurkat T cell line.

The cytoplasmic tail of ICAM-R has homology with a motif that has beenidentified in the cytoplasmic domains of some T cell antigen receptorsubunits, B cell membrane immunoglobulin antigen receptor subunits andmast cell Fc receptor subunits Reth, M., Nature, 338:383-384 (1989)!.The motif (see below) is known by various acronyms, including antigenreceptor activation motif (ARAM), T cell activation motif (TAM) andantigen receptor homology 1 (ARH 1). Since its identification, thismotif has been demonstrated to be necessary and sufficient for thetransduction of signals from the membrane. ##STR1##

Shown is the consensus motif (top line) aligned with the ICAM-Rcytoplasmic tail residues 483-501. Residues that are homologous with theconsensus are underlined. The asterisk indicates the position of thestop codon in the truncated protein haCTΔ₂₈₆₋₄₉₃.

To determine what region of the ICAM-R tail consensus motif that mightbe required for signaling in T cells, two cytoplasmic domain truncationshave been created from haWT₂₈₆₋₅₈₆. These are truncated at residues 505(haCTΔ₂₈₆₋₅₀₅) and 493 (haCTΔ₂₈₆₋₄₉₃) thus dividing the cytoplasmicdomain roughly into thirds. The haCTΔ₂₈₆₋₅₀₅ protein leaves theconsensus motif intact but removes the carboxyl terminal 13 residues ofthe native protein, while haCTΔ₂₈₆₋₄₉₃ divides the motif in half,leaving only one of the tyrosine residues in the motif present. Thesetruncations have been subcloned into the expression vector pMHneo foranalysis in the Jurkat T leukemic cell line.

B. Jurkat Cell Expression

Jurkat cells were grown in RPMI 1640 medium, 10% FBS supplemented withpenicillin/streptomycin and L-glutamine (RPMI complete) under standardcell culture conditions. For each electroporation condition, 5×10⁶Jurkat cells at midphase of logarithmic growth were pelleted and rinsedin PBS-D, pelleted again and resuspended in PBS-D to a density of 1×10⁷/ml. One half of one ml of suspended cells was transferred to a sterilecuvette (0.4 cm electrode gap) and 20 ug of linear plasmid DNA wereadded, mixed gently and incubated on ice for 10 minutes. A sufficientquantity of DNA was linearized by BglII digestion and prepared asfollows. The linear DNA was extracted once with phenol/chloroform andprecipitated with ethanol. After a 70% EtOH rinse and lyophilization,the DNA was resuspended in PBS-D to 1 mg/ml. The DNA/cell mix was thensubjected to a 0.625 V/cm electric field with a pulse capacitance of 960uFd. After a 10 minute incubation on ice, the cells were placed on 4 mlmedium and allowed to replicate at 37° C. to allow for integration ofthe plasmid DNA. After 48 hours, the entire cell population from eachelectroporation condition was plated out into 96 well plates such that1-3×10⁴ cells were plated into each well in medium supplemented with1.25 mg/ml G418 (Life Technologies, Bethesda, Md.). After approximatelyfour weeks of drug selection, sufficient numbers of wells had cellsgrown from partial to complete confluency. These cells, and subsequentones that grew, were routinely screened by FACS for positive stainingwith the HA epitope tag antibody, 12CA5. Positive wells were expandedand restained for surface expression of the following proteins andcontrols: CD3 (OKT3 antibody), ICAM-R (ICR-1.1 antibody), HA (12CA5antibody), IgG 2a and 2b matched controls and FITC-conjugated secondaryalone. Those wells that were positive for CD3, ICAM-R and HA and werenegative for the isotype controls and the secondary antibody detectingreagent alone were expanded and FACS sorted to retain cells exhibitingthe highest expression levels of the HA antigens.

C. Co-Stimulation and Cytokine Release

Wells of a 96 well plate (Immulon 4, Dynatech) were coated with 50 ul ofa 1 ug/ml OKT3 monoclonal antibody in PBS-D for 16 hours at 4° C. Thisdose of antibody alone provides minimal signaling for IL-2 release. TheOKT3 treatment was removed and replaced with buffer alone or anti-ICAM-R(ICR-1.1) (10 ug/ml in PBS-D) and incubated at 37° C. for at least 2hours. Monoclonal antibody coating was done in replicates of two or morewells and pooled to provide sufficient quantities of conditioned mediafor ELISA and/or bioassay. Two hundred fifty thousand Jurkat cells ortransfectants thereof, were placed into antibody coated wells or buffercoated negative control wells in 0.1 ml of RPMI complete medium.Following incubation of about 16-24 hrs at 37° C. in a humidifiedatmosphere containing 5% CO₂, the medium was transferred to a fresh 96well round bottom plate, spun to pellet cells carried over andtransferred to a fresh plate. Samples were frozen and stored at -70° C.IL-2 ELISAs were performed using commercially available kits (BiosourceIntl. Co., Camarille, Calif.) by making serial dilutions of the samples.

Expression of the haWT₂₈₆₋₅₁₈ and haCTΔ₂₈₆₋₄₈₄ had differing effects onthe ability of the cells to be costimulated by ICR-1.1. Expression ofhaWT₂₈₆₋₅₁₈ inhibited by about 60% the co-stimulatory response deliveredvia ICAM-R as compared to the response from cells expressinghaCTΔ₂₈₆₋₄₈₄ or vector transfectants alone. A conclusion of theseexperiments is that intracellular signaling and modulation of the IL-2response through ICAM-R requires an intact cytoplasmic domain.Furthermore, this result implies that this system can be used to definethe critical amino acid residues in ICAM-R by introducing the mutationsoutlined above and in Table 12.

D. Associate Protein p23

When Jurkat transfectants expressing haWT₂₈₆₋₅₁₈ and haCTΔ₂₈₆₋₄₈₄ weremetabolically labeled with ³⁵ S! methionine, lysed andimmunoprecipitated using anti-HA antisera, proteins of approximately 45and 40 kD were visualized. Under reduced conditions, both of thetransfectants yielded immune complexes which also contained anassociated protein which migrated in SDS-PAGE at approximately 23kD(p23). Under non-reduced conditions, the 23 kD protein band apparentlyshifted mobility and formed a complex with haWT₂₈₆₋₅₁₈ or haCTΔ₂₈₁₋₄₈₄of 68 and 60 kD respectively. In addition, higher molecular weightcomplexes were visualized in haWT₂₈₆₋₅₁₈ lysates of approximately 100 kDwhich may be homodimers of the protein or heteromeric complexes.

When unlabeled Jurkat transfectants were immunoprecipitated with anti-HAserum, analyzed by SDS-PAGE and transferred to nitrocellulose, only thefull length haWT₂₈₆₋₅₁₈ and haCTΔ₂₈₆₋₄₈₄ proteins were visualized whenthe membrane was probed with anti HA monoclonal antibody. In addition,nothing was visualized when an antisera that recognizes the ICAM-Rcytoplasmic tail was used to probe the membrane. Therefore, it isunlikely that p23 is a proteolytic degradation product of thehaWT₂₈₆₋₅₁₈ and baCTΔ₂₈₆₋₄₈₄ proteins.

E. Expression of ICAM-R Cytoplasmic Tail Variants in an ICAM-R DeficientJurkat T Cell Line

To study the structure/function of the cytoplasmic domain of ICAM-R, adeletion analysis was initiated as described in Example 22, section A.These constructs were designed to synthesize progressive cytoplasmicdomain deletions associated with an extracellular domain consisting ofthe HA (hemagglutinin) epitope tag fused to c-type Ig domains 4 and 5from ICAM-R. Over-expression of these proteins in Jurkat cells thatexpress endogenous ICAM-R was thought to lead to competition, such thatcoupling to cytoplasmic signaling molecules via endogenous ICAM-R mightbe abrogated. An additional strategy was devised in which a Jurkat cellline deficient in endogenous ICAM-R expression was developed and ICAM-Rcytoplasmic domain deletions/point mutants could be expressed to lookfor functional coupling.

Jurkat cells (subline J77, from S. J. Burakoff, Boston, Mass.) weregrown as described in Example 22, Section B above. Cells were stainedfor flow cytometry using ICR-1.1 and 9.2 (non-competitive bindingmonoclonal antibodies) and indirect FITC conjugate detection understerile conditions. The final washed pellet was sorted, such that theICR-1.1 and 9.2 deficient cells (˜3% of input cells) were collected. Therecovered cells were expanded and re-sorted as described such thatgreater than 99% were deficient in ICR-1.1 and 9.2 binding. These cellswere designated J77.50.3.

A construct was generated such that the coding sequence for the ICAM-Rsignal peptide preceded the HA tag, which was followed by the remainderof the coding sequence for ICAM-R. This construct was subcloned intopMHneo Hahn et al., Gene, 127: 267-268 (1993)! and designated haFL.Three successive carboxy terminal deletions were constructed from theplasmid, such that the respective termini were residues 505 (haCTΔ505),493 (haCTΔ493) and 484 (haCTΔ484). In addition, selected point mutantsof residues within the predicted cytoplasmic domain were generated.These mutants were designed to study the role of tyrosine residues inthe biology of ICAM-R. The following point mutants were generated:Tyr479Phe, Tyr490Phe, Tyr498Phe, Ser487Ala, Ser489Ala, Ser503Ala andSer515Ala. The plasmids were electroporated into J77.50.3 cells, stabledrug resistant lines were selected and surface expression determined byindirect flow cytometry.

Experiments were performed using two independently isolated cell linesfor each plasmid construct or the vector alone. Stable lines expressingsimilar levels of ICAM-R variants, as determined by indirect flowcytometry, were chosen for functional studies. Cells were co-stimulatedwith plate immobilized monoclonal antibodies ICR-1.1 and OKT3, and IL-2release from the lines was quantitated by ELISA as previously describedin Example 24C. Results were expressed as fold increase of IL-2 releasedfrom cells plated onto co-immobilized ICR-1.1 (10 μg/ml) plus OKt3 (1μg/ml) divided by release from OKT3 (10 μg/ml) alone. The full lengthICAM-R protein (haFL) gave 3.1 fold induction. The deletions gave thefollowing fold inductions: haCT 505 induction was 1.5 fold, haCT 493induction was 1.3 fold, haCT 484 was 0.8 fold. Vector control inductionwas 0.7 fold. These results demonstrate that deletion of the cytoplasmicdomain abrogates the capability of ICAM-R to co-stimulate Jurkat cells.In addition, removal of the residues 505-518 leads to the mostsignificant reduction in co-stimulatory responsiveness. This impliesthat residues 505-518 play a relatively larger role in the response ascompared to residues 484-505.

To ascertain additional functional consequences of ICAM-R carboxylterminal deletions, the Jurkat cell lines expressing the ICAM-R variantsdescribed above were tested for the ability to adhere and spread onantibody coated plastic or to undergo antibody-mediated aggregation.Bacterial petri plates were coated with 50 μl drops of ICR-1.1 or w6/32(10 μg/ml) (anti-MHC class I isotype matched control monoclonalantibody) in PBS for 120 minutes at 37° C. The plastic was rinsed twiceprior to spotting 50 μl drops of J77.50.3 cell lines. Duplicate celllines expressing these proteins at equivalent levels were tested. After20 minutes at 37° C. the plate was flooded with 2% glutaraldehyde in PBSand incubated for 30 minutes to fix adherent cells. Fixed cells wererinsed twice with PBS and scored visually for the ability to flatten andspread. Cells expressing either haFL, haCTΔ505, haCTΔ493, Tyr479Phe orTyr498Phe spread onto the ICR-1.1 coated plastic surface and not ontothe w6/32 coated surface. Cells expressing haCTΔ484 or Tyr490Phe wereincapable of flattening down onto the ICR-1.1 coated surface. Weyrich etal., J. Clin. Invest., 95: 2297-2303 (1995) recently described theeffect of a ICR-1.1 coated plastic surface on normal monocytes fromperipheral blood. After two hours, these cells flattened down and spreadwith a similar overall morphology to that seen with the haFL expressingJ77.50.3 cell line. These results imply that engagement of ICAM-R bydomain 1 specific monoclonal antibodies induces similar dramaticmorphologic changes for different cell types expressing ICAM-R. Inaddition, the deletion analysis suggests that residues 484-505 arerequired for this phenomenon. Interestingly, changing the charge locallyin this region by addition of a less polar residue (Tyr to Phe atposition 490) suggests that the structure of this domain is importantfor spreading competence.

Selected monoclonal antibodies against ICAM-R have been shown to inducehomotypic aggregation (Example 19). One of these monoclonal antibodies(ICR-1.1) was used to induce aggregation of the J77.50.3 cell linesexpressing either full length ICAM-R (haFL) or carboxy terminal variantsof ICAM-R. ICR-1.1 was added to 200,000 cells at 10 μg/ml in 96 wellplates and incubated 37° C. At 1 and 24 hours after incubation the wellswere visually screened for aggregates. Lines expressing either haFL,haCT 505, haCTΔ493, Tyr479Phe or Tyr498Phe were aggregation competent.Cells expressing haCTΔ484 or Tyr490Phe were incapable of forminginducible aggregates.

EXAMPLE 25

The ICAM-R binding site for LFA-1 was localized to the amino terminaldomain (domain 1) of ICAM-R. In addition, specific residues in domain 1involved in the interaction of ICAM-R with LFA-1 were identified.

A. Production of ICAM-R Immunoglobulin Chimeras

The entire extracellular coding region of ICAM-R (nucleotides 1 to 1470comprising the leader peptide sequence and all five Ig-like domains)isolated from pVZ147 (Example 4) by PCR was ligated in frame with a DNAfragment encoding the hinge and CH2 and CH3 coding regions of human IgG₁antibody. The resulting chimeric construct, ICAM-R/IgG, was expressedfrom the CMV promoter in the plasmid pcDNA1/Amp (Invitrogen). VariantICAM-R/IgG fusion proteins were also produced from expression constructsin which either ICAM-R domain 1 or domain 3 encoding sequences had beendeleted or in which mutations encoding amino acid substitutions had beenintroduced by site directed mutagenesis.

COS cells were transfected with the expression constructs by theDEAE-dextran method and the culture supernatant was collected. Fusionprotein was purified from the culture supernatants using a Protein-Aaffinity (Prosep-A, Bioprocessing Ltd., England) column. A 1 ml bedvolume Prosep-A column was equilibrated with PBS and the culturesupernatant was loaded by gravity flow. The column was serially washedwith 10 ml of 0.5 M diethanolamine and 10 ml of 0.05 M citric acid pH5.0 to remove weakly binding proteins. Subsequently the ICAM-3/IgGfusion protein (or variant fusion protein) was eluted with 0.05 M citricacid pH 3.0. The eluate was neutralized with one-sixth volume of 1.5 MTris pH 9.0. SDS-PAGE analysis of the protein revealed a band of about225 kD in size which migrated to about 110 kD size under reducingcondition indicating that the secreted fusion protein is a dimer.

The purified wild type ICAM-R/IgG chimera was quantitated by Micro BCAreagent (Pierce, Rockford, Ill.) according to the manufacturer'sinstructions. An estimate of the concentration of variant ICAM-R/IgGproteins was determined by coating plastic microtiter wells with serialdilutions of mutant ICAM-R chimeras or highly purified (>95%) ICAM-R/IgGfollowed by detection with horseradish peroxidase (HRP)-conjugated goatanti-human IgG (Fc-specific) antibody.

B. Binding of JY Cells to Wild Type ICAM-R/IgG and ICAM-R DomainDeletion/IgG Chimeras

The binding of the lymphoblastoid cell line JY, which expresses LFA-1,to wild type ICAM-R/IgG chimera was examined in the presence and absenceof LFA-1 specific antibodies or antibodies specific for either domain 1or 2 of ICAM-R. ICAM-R chimeras were diluted to 10 μg/ml in 0.1 MNa-carbonate/bicarbonate buffer pH 9.6 and used to coat triplicate wells(50 μl/well) of an Immulon 4 96-well plate (Dynatech) overnight at 4° C.Wells were washed three times in PBS and blocked with 1% BSA (in PBS)for 1 hour at 37° C. JY cells were labeled with the fluorescent dyeCalcein (Molecular Probes, Eugene, Oreg.) at 8 μg/ml at 37° C. in serumfree RPMI for 20 minutes. Cells were washed with RPMI and resuspended inbinding buffer (0.2% HSA in RPMI). Approximately 1×10⁵ cells were addedto each well containing the binding buffer with or without antibody (20μg/ml of ICR-2.1, ICR-1.1, ICR-3.1, ICR-5. 1, ICR-7.1, ICR-8.1, ICR-4.2,ICR-6.2, ICR-9.2, TS1/22 or 60.3) so that the final volume was 0.35 ml.Plates were incubated at 37° C. in the dark for 45 minutes and inputfluorescence was quantitated with a fluorescence reader (Millipore) in a96-well format. Unbound cells were removed by inverting the plate in 0.1% BSA in PBS for 20 minutes. Bound cells were quantitated by measuringthe remaining fluorescence and presented as percent input minusbackground binding to wells coated with BSA alone.

Adhesion of the JY cells to the plate-bound ICAM-R/IgG chimera waspredominantly LFA-1 dependent as shown by the ability of the LFA-1specific monoclonal antibody TS 1/22 to completely block binding. SixICAM-R monoclonal antibodies also blocked the adhesion of JY cells toICAM-R to varying extents. ICR-2.1 inhibited about 85% of JY cellbinding. Five others ICR-1.1, 3.1, 5.1, 7.1 and 8.1 inhibited JY celladhesion approximately 60 to 80%. Three monoclonal antibodies ICR-4.2,6.2 and 9.2 had no appreciable effect on the adhesion of JY cells toICAM-R. These results suggest that LFA-1 interacts with ICAM-R domain 1because monoclonal antibodies specific for ICAM-R domain 1 but notdomain 2 block binding.

Binding of JY cells to domain deletion chimeras was also tested in theforegoing assay. Deletion of domain 1 resulted in complete loss of theability of ICAM-R to promote LFA-1 mediated cell adhesion whereasdeletion of domain 3 had essentially no effect. Collectively, from theseresults and the monoclonal antibody blocking results, it is apparentthat ICAM-R interacts with LFA-1 primarily through its amino terminaldomain 1.

C. Binding of JY Cells to Amino Acid Substituted ICAM-R/IgG Chimeras

ICAM-R/IgG chimeras with single or double amino acid substitutions indomain 1 were also tested in the binding assay described in Section Babove. The mutation, E32K/AS (wherein mutation nomenclature is the sameas in Example 14) resulted in a significant decrease in LFA-1 binding.In addition, the mutations E37T/AS and Q75I/AS nearly abolished adhesionof JY cells. To determine the contribution of individual residues wegenerated two additional mutations, E37/A and T38/A. The E37/A mutationcompletely abrogated adhesion of JY cells to ICAM-R. The T38/A mutationalso resulted in a significant (70-80%) reduction in cell adhesion.These residues are displayed in a model of ICAM-R domain 1 along withtheir effect on LFA-1 binding in FIG. 16, wherein β strands (widearrows) were based on secondary structure predictions of ICAM-R andICAM-1 as well as on alignment with the tenth type 3 repeat offibronectin. Similar to the epitopes mapped for blocking ICAM-R specificmonoclonal antibodies, residues implicated in LFA-1 binding locate toone face of domain 1 in this model.

Because carbohydrates are known to influence the ligand bindingproperties of several cell adhesion molecules, the effects of N-linkedglycosylation of ICAM-R on LFA-1 binding were determined. In domain 1 ofICAM-R there are five potential N-linked glycosylation sites. Of thesesites, N71 and N82 are closest to the residue Q75I which is shown aboveto be involved in LFA-1 binding. Replacement of N71 or N82 withglutamine did not significantly alter the adhesive properties of ICAM-R.

Moreover, disruption of ICAM-R domain 2 structure did not decrease LFA-lbinding. A domain 2 mutation L121/P resulted in significant loss of theepitopes for all three domain 2 antibodies. When the L121/P mutantchimera was tested for capacity to support cell adhesion, wild typelevels of LFA-1 mediated binding were observed.

EXAMPLE 26

Experiments were performed that show that another leukocyte integrinα_(d) /CD18 is also a ligand for ICAM-R. The leukocyte intergrin α_(d)is described in co-pending, co-owned U.S. patent application Ser. No.08/173,497 and in co-owned, concurrently filed U.S. patent applicationSer. No. 08/173,497.

A. Human α_(d) binds to ICAM-R in a CD18-dependent fashion

In replicate assays, soluble ICAM-1, ICAM-R, or VCAM-1 IgG1 chimericfusion proteins were immobilized on plastic and the ability of α_(d)/CD18 or LFA-1 transfected CHO cells (see co-owned, concurrently filedU.S. patent application Ser. No. 08/286,889 identified as attorneydocket No. 32168) to bind the immobilized ligand was determined.Transfected cells were labeled internally with calcein, washed inbinding buffer (RPMI with 1% BSA), and incubated in either buffer only(with or without 10 ng/ml PMA) or buffer with anti-CD18 at 10 μg/ml.Transfected cells were added to 96-well Immulon 4 microtiter platespreviously coated with soluble ICAM-1/Ig, ICAM-R/Ig or VCAM-1/Igchimera, or bovine serum albumin (BSA) as a negative control. Wells wereblocked with 1% BSA in PBS prior to addition of labeled cells. Afterwashing the plates by immersion in PBS with 0.1% BSA for 20 minutes,total fluorescence remaining in each well was measured using a Cytofluor2300 (Millipore, Milford, Mass.).

In experiments with immobilized ICAMs, α_(d/CD) 18 co-transfectantsconsistently showed a 3-5 fold increase in binding to ICAM-R/IgG wellsover BSA coated wells. The specificity and CD18-dependence of thisbinding was demonstrated by the inhibitory effects of anti-CD18 antibodyTS1/18. The binding of cells transfected with LFA-1 to ICAM-1/IgG wellswas comparable to the binding observed with BSA coated wells. LFA-1transfected cells showed a 2-3 fold increase in binding to ICAM-1/IgGwells only following pretreatment with PMA. PMA treatment of α_(d) /CD18transfectants did not affect binding to ICAM-1/IgG or ICAM-R/IgG wells.No detectable binding of α_(d) /CD18 transfectants to VCAM-1/IgG wellswas observed.

Binding of α_(d) /CD18-transfected cells to soluble ICAM-1/IgG,ICAM-R/IgG, or VCAM-1/IgG chimeras was determined by flow cytometry.Approximately one million α_(d) /CD18-transfected CHO cells (grown inspinner flasks for higher expression) per measurement were suspended in100 μl binding buffer (RPMI and 1% BSA) with or without 10 μg/mlanti-CD18 antibody. After a 20 minute incubation at room temperature,the cells were washed in binding buffer and soluble ICAM-1/IgG orICAM-R/IgG chimera was added to a final concentration of 5 μg/ml.Binding was allowed to proceed for 30 minute at 37° C., after which thecells were washed three times and resuspended in 100 μl binding buffercontaining FITC-conjugated sheep anti-human IgGI at a 1:100 dilution.After a 30 minute incubation, samples were washed three times andsuspended in 200 μl binding buffer for analysis with a Becton DickinsonFACScan.

Approximately 40-50% of the ad/CD18 transfectants indicated binding toICAM-R/IgG, but no binding to ICAM-1/IgG or VCAM-1/IgG chimericproteins. Pretreatment of transfected cells with PMA has no effect onα_(d) /CD18 binding to either ICAM-1/IgG or ICAM-R/IgG. Binding byICAM-R was reduced to background levels after treatment of α_(d) /CD18transfectants with anti-CD18 antibody TS1/18. Consistent with theimmobilized adhesion assay, PMA treatment of transfected cells had noaffect on α_(d) /CD18 interaction with ICAM-R/Ig.

The collective data from these two binding assays illustrate thatα_(d/CD) 18 binds to ICAM-R and does so preferentially as compared toICAM-1 and VCAM-1. The α_(d) /CD18 binding preference for ICAM-R overICAM-1 is opposite that observed with LFA-1 and Mac-1. Thus modulationof α_(d) /CD18 binding may be expected to selectively affect normal andpathologic immune function where ICAM-R plays a prominent role.Moreover, results of similar assays, in which antibodies immunospecificfor various extracellular domains of ICAM-R were tested for theirability to inhibit binding of ICAM-R to α_(d) /CD18 transfectants,indicated that α_(d) /CD18 and LFA-1 interact with different domains ofICAM-R.

The failure of LFA-1 to bind ICAM-1/IgG or ICAM-R/IgG in solutionsuggests that the affinity of binding between LFA-1 and ICAM-1 or ICAM-Ris too low to permit binding in solution. Detection of α_(d) /CD18binding to soluble ICAM-R, however, suggests an unusually high bindingaffinity.

EXAMPLE 27

ICAM-R was also determined to interact with the β₁ integrin VLA-4 (α₄/β₁). ICAM-R domain specific monoclonal antibodies and an ICAM-R domaindeletion IgG chimera were used to map the VLA-4 binding to a site inICAM-R domains 3-5. VLA-4 is expressed on all leukocytes with theexception of neutrophils. VLA-4 is also expressed on non-hematopoieticcells, including fibroblasts and neural crest cells. VLA-4 monoclonalantibodies inhibit the binding of leukocytes to cytokine activatedendothelium Elices et al., Cell, 60:577-584 (1990)!, lymphocytehomotypic aggregation Pulido et al., J. Biol. Chem., 266:10241-10245(1991)! and cytotoxic T cell mediated killing Clayberger et al., J.Immunol., 138:1510-1514 (1987)!. Adhesion of VLA-4 positive leukocytesto endothelium has been implicated in the process of atherogenesisCybulsky et al., Science, 251:788-791, (1991)!, encephalomyelitis Baronet al., J. Exp. Med., 177:57-68, (1993)!, allogeneic graft rejectionPelletier et al., J. Immunol, 149:2473-2481 (1992)! and rheumatoidarthritis Morales-Ducret et al., J. Immunol., 149:1424-1431 (1992)!.

A. Binding of Jurkat Cells to Plate-Bound ICAM-R

shICAM-R (Example 9) was diluted to 10 μg/ml in 50 mM carbonate bufferpH 9.6. Fifty μl/well of this dilution was used to coat wells of a96-well plate (Dynatech) by incubation for 16 hours at 4° C. Prior toadhesion assay, the wells were emptied and blocked for 1 hour at 37° C.with 1% BSA (Cohn Fraction V, Sigma) in PBS. Jurkat cells were labeledwith 8 μg/ml Calcein AM (Molecular Probes) in serum free culture mediumfor 20 minutes at 37° C. Rinsed cells were treated with α₄ or β₁specific monoclonal antibody, and 1.3×10⁵ cells were distributed perwell in RPMI, 0.2% HSA (Calbiochem). After a 50 minute incubation at 37°C., total fluorescence per well was determined using a Cytofluor 2300(Millipore), then the plate was inverted in 37° C. PBS, 1% BSA for 30minutes. The plate was removed and about 100 μl/well aspirated using a12 place manifold to remove cells in suspension near the mouth of thewells. The remaining fluorescence was determined and percent boundcalculated from triplicate wells for each condition tested.

The α₄ specific monoclonal antibodies IC/A4.1 (ICOS Corp., Bothell,Wash.), 163H (anti-CD49d obtained from Dr. Michael Longenecker,University of Alberta, Edmonton, Canada) and HP2/1 (anti-CD49d, AMAC,Westbrook, Me.) and the β₁ specific monoclonal antibodies K20(anti-CD29, AMAC) and 3S3 (anti-CD29 obtained from Dr. John Wilkins,University of Manitoba, Winnepeg, Canada) blocked adhesion of Jurkatcells. Since Fab fragments of the β₁ monoclonal 3S3 also blockedadhesion, inhibition does not appear to be a consequence of signalstransduced following an antibody-induced antigen crosslinking. Theability of multiple α₄ and β₁ specific monoclonal antibodies to blockVLA-4 binding to ICAM-R suggests a direct interaction between VLA-4 andICAM-R. In contrast, these α₄ and β₁ antibodies did not block theCD18-dependent JY cell binding to ICAM-R.

B. Localization of the VLA-4 Binding Site

The VLA-4 binding site on ICAM-R was localized to domains 3-5 bycomparing Jurkat and JY cell binding to wild type and domain deletionICAM-R/IgG chimeras (Example 25) in the presence or absence of domainspecific monoclonal antibodies to ICAM-R. The domain deletion ICAM-R/IgGchimera included ICAM-R domains 3, 4 and 5 and thus lacked the LFA-1binding site within ICAM-R domain 1. Adhesion assays were performedaccording to the procedure described in Example 26.

The binding of Jurkat and JY cells to the ICAM-R/IgG chimeras wascompared to the binding results for soluble ICAM-R. Jurkat cells boundat equivalent levels to all three forms of ICAM-R and was VLA-4dependent. ICAM-R antibody ICR-19.3 specific for domain 3 completelyblocked Jurkat cell binding to all forms of ICAM-R. In contrast, JYcells did not bind to domain deletion ICAM-R/IgG chimera lacking theLFA-1 binding site in domain 1. Monoclonal antibody specific for ICAM-Rdomain 1 did not block binding of Jurkat cells to wild-type ICAM-R.

The presence of three integrin binding sites (for LFA-1, α_(d) /CD18 andVLA-4) on ICAM-R may have several functional implications. Integrinbinding to different sites on ICAM-R may transduce distinctintracellular signals. In addition, if LFA-1, α_(d) /CD18 and VLA-4could bind simultaneously to ICAM-R, distinct or synergistic signals maybe transduced through VLA-4 or LFA-1 in an opposing cell.

C. Inhibition of T Cell Activation by ICAM-R Binding to VLA-4

In previous examples, the effects on responding cells of engagement ofICAM-R by specific monoclonal antibodies was determined. In thissection, the effect of engagement of the integrin receptors LFA-1 andVLA-4 by rICAM-R protein was measured.

T cell costimulation by recombinant ICAM-R and CD3 antibody in thepresence of monoclonal antibodies which block ICAM-R adhesion to VLA-4was measured (Example 20F). The monoclonal antibodies tested werespecific for domain 3 of ICAM-R or the α₄ subunit of VLA-4. All of theblocking monoclonal antibodies dramatically enhanced the proliferativeresponse to immobilized CD3 antibody. These results suggest that ICAM-Rbinding to VLA-4 inhibits T cell activation.

ICAM-R/IgG chimeras that preferentially bind LFA-1 or VLA-4 were alsoused to demonstrate the differential effects of ICAM-3 interaction witheither receptor on T-cell activation. An ICAM-R/IgG variant E37T-Igwhich binds VLA-4 but not LFA-1, and an ICAM-R/IgG variant D231H-Igwhich demonstrated reduced VLA-4 binding capacity while retaining fullLFA-1 binding capacity, were prepared by methods similar to thosedescribed in Example 25. Both variants were tested for effects on T-cellproliferation in response to CD3 mAb. In comparison to the HSA control,wild-type ICAM-3/IgG and variant D231H-Ig enhanced CD3 mAb driven T-cellproliferation. Similar levels of costimulation were induced by the LFA-1binding D231H-Ig or wild-type ICAM-R/Ig chimera which binds both LFA-1and VLA-4. This result supports the concept that LFA-1 represents thedominant functional T-cell integrin receptor for ICAM-R in this model. Acontrasting effect on T-cell proliferation was observed in culturescontaining the selective VLA-4 binding variant E37T-Ig. In comparison tothe HSA control, E37T-Ig markedly inhibited T-cell proliferation inresponse to CD3 mAb.

Similar results were obtained from assays of IL-2 production in responseto costimulation by anti-CD3 monoclonal antibody and shICAM-R or mutantICAM-R/IgG chimeras. shICAM-3, wild type ICAM-R/IgG chimera and to alesser extent D231H-Ig enhanced IL-2 production in comparison to the HSAcontrol. In contrast the VLA-4 binding mutant E37T-Ig inhibited IL-2production in response to CD3 mAb. These results demonstrate that ICAM-Relicits contrasting effects on T-cell function by interacting withdistinct T-cell integrins.

Since rICAM-R in soluble form is of sufficient affinity to bindintegrins such as α_(d) /CD18 (see Example 26), these results aresignificant since it is collectively implied that variant solubleICAM-Rs may be utilized therapeutically.

Example 28

Circulating forms of ICAM-R (cICAM-R) were identified in human serum.Using a sandwich-ELISA with two monoclonal anti-ICAM-R antibodies(ICR-4.2 and ICR-8.1), cICAM-R was found in concentrations between 40 to360 ng/ml in all of 112 healthy controls. An analysis of patient serafrom ten different immune-mediated diseases revealed a distinct patternof expression. Significantly elevated cICAM-R levels were found inrheumatoid arthritis, systemic lupus erythematosus, Guillain-Barresyndrome and multiple sclerosis, but not in type I diabetes, Grave'sdisease, chronic autoimmune thyroiditis, ulcerative colitis or Crohn'sdisease. cICAM-R levels were significantly higher in lupus patients withactive compared to non-active disease. There was no uniform increase ofcICAM-R levels in chromic inflammatory/autoimmune conditions. Serumlevels of cICAM-R did not correlate with cICAM-1 concentrations ineither control samples or in patients. The majority of patients hadeither elevated cICAM-R or cICAM-1 levels, but not both.

A circulating form of ICAM-R is thus present in human sera. cICAM-Rexpression is elevated in certain immune-mediated disease states butoccurs independently of cICAM-1.

EXAMPLE 29

Clearly, polynucleotides (e.g., DNA and RNA) encoding ICAM-R are usefulnot only in securing expression of ICAM-R and variant polypeptides; theymay readily be employed to identify cells (especially cells involved inimmunological processes) which express ICAM-R in a normal or activatedstate. Typical detection assays involving ICAM-R DNA include Northernblot hybridization, RNAse protection, and in situ hybridizationcytological assays wherein the DNA or RNA (in suitably labelled,detectable form) hybridizes to RNA in the sample. ICAM-R encoding DNA(especially DNA encoding the first, fourth and fifth domains which haveless homology to DNAs encoding ICAM-1 and ICAM-2 than the DNAs encodingdomains 2 and 3) is expected to be useful in isolating genomic DNAencoding ICAM-R including genomic DNA specifying endogenous expressioncontrol DNA sequences for ICAM-R DNA. As previously noted, knowledge ofpolynucleotide sequences encoding ICAM-R and/or controlling expressionof ICAM-R makes available a variety of antisense polynucleotides usefulin regulating expression of ICAM-R.

The present invention makes available the production of ICAM-Rpolypeptides and variants thereof, especially including solublefragments thereof, such as fragments comprising one or more of the fiveimmunoglobulin-like domains of ICAM-R in glycosylated, non-glycosylated,or de-glycosylated forms. Pharmaceutical compositions including theprotein products of the invention have therapeutic potential in themodulation of immune cell activation/proliferation, e.g., as competitiveinhibitors or stimulatory agents of intercellular and intracellularligand/receptor binding reactions involving ICAM-R. Such therapeuticpotential is especially projected for "immunoadhesin" type recombinanthybrid fusion proteins containing, at their amino terminal, one or moredomains of ICAM-R and, at their carboxy terminal, at least one constantdomain of an immunoglobulin. Such hybrid fusion proteins are likely tobe available in the form of homodimers wherein the Ig portion providesfor longer serum half life and the ICAM-R portion has greater affinityfor the ICAM-R binding partner than ICAM-R itself. Other multimericforms of ICAM-R which may have enhanced avidity are also projected tohave therapeutic potential.

Antibody substances and binding proteins, especially monospecificantibodies including monoclonal and polyclonal antibodies, are madereadily available by the present invention through the use of immunogenscomprising cells naturally expressing ICAM-R, recombinant host cellsproducing polypeptide products of the invention, the ICAM-R polypeptideproducts themselves, and polypeptide products of the invention bound toan ICAM-R specific antibody that stimulates cell-cell aggregation (i.e.,polypeptide products that may be in a "high affinity" bindingconformation). Such antibodies and other ICAM-R specific bindingproteins can be employed for immunopurification of ICAM-R and variantsand in pharmaceutical compositions for therapies premised on blockingand/or stimulating the ligand/receptor binding of ICAM-R and solublefragments thereof. For use in pharmaceutical compositions, ICAM-Rspecific antibody and anti-idiotypic antibody substances may behumanized (e.g., CDR-grafted) by recombinant techniques well-known inthe art. As illustrated in the foregoing examples, antibodies todistinct regions of ICAM-R may be employed to block adhesiveinteractions mediated by distinct integrins (e.g., LFA-1, VLA-4 andα_(d) /CD18). Also, antibodies specific for distinct regions of ICAM-Rmay be employed in ELISA systems involving immunological "sandwiches"for monitoring inflammatory processes characterized by increases inamounts of soluble ICAM-R polypeptides in body fluids such as serum. Asoutlined in Example 28, it is anticipated that such monitoring of ICAM-Rlevels as a surrogate marker of disease progression will be particularlyuseful in syndromes such as systemic lupus erythematosus, rheumatoidarthritis, multiple sclerosis and Guillan-Barre syndrome and may beuseful as an early predictor of the onset of clinical episodes so thattherapeutic drugs can be applied in a more timely fashion. As well, theonset of syndromes such as preterm labor, which may be mediated in partthrough an inflammatory process, may also be monitored by assessinglevels of circulating ICAM-R in body fluids.

Inflammatory conditions which may be treated or monitored with ICAM-Rrelated products include conditions resulting from a response of thenon-specific immune system in a mammal (e.g., adult respiratory distresssyndrome, multiple organ injury syndrome secondary to septicemia,multiple organ injury syndrome secondary to trauma, reperfusion injuryof tissue, acute glomerulonephritis, reactive arthritis, dermatosis withacute inflammatory components, stroke, thermal injury, hemodialysis,leukapheresis, ulcerative colitis, Crohn's disease, necrotizingenterocolitis, granulocyte transfusion associated syndrome,atherosclerosis and cytokine-induced toxicity) and conditions resultingfrom a response of the specific immune system in a mammal (e.g.,psoriasis, organ/tissue transplant rejection and autoimmune diseasesincluding Raynaud's syndrome, autoimmune thyroiditis, EAE, multiplesclerosis, rheumatoid arthritis, diabetes, and lupus erythematosus).ICAM-R products of the invention may also be useful in monitoring andtreating asthma, tumor growth and/or metastasis, and viral infection(e.g., HIV infection).

In particular, it is anticipated that disease processes in which T cellactivation plays a central and essential triggering role will beimpacted beneficially by products of the invention described herein.This inference is drawn in part from the findings outlined in Example 20wherein monoclonal antibodies specific to ICAM-R and recombinant formsof ICAM-R protein were shown to modulate the response of human Tlymphocytes to activating stimuli. Moreover, the therapeutic use ofICAM-R analogs incorporating specific amino acid substitutions (e.g.E37T or D231H) chosen to enhance or diminish their specificimmunomodulatory properties (see Example 27) are expected to be usefulin this regard. Since analogs of ICAM-R expressed as chimeric fusionswith human immunoglobulin constant regions were shown to bind at leastone integrin, it is anticipated that administration of these moleculesin soluble form will be therapeutically useful. Specific examples of Tcell dependent diseases for which ICAM-R related products areanticipated to have utility include but are not limited to asthma,psoriasis, diabetes, graft vs. host disease, tissue transplantrejection, and multiple sclerosis.

As illustrated in the foregoing examples, products of the invention canalso be used to modulate the biological responses of monocytic cells andadhesion mediated by at least one integrin expressed selectively bymacrophages, α_(d). Thus, diseases wherein macrophages play a centralgenerative role are also expected to benefit from products of theinvention. For example, the formation of atherosclerotic plaques both asoccurs progressively over time in humans and also as a consequence ofsolid organ or vessel engraftment (e.g., coronary bypass surgery)involves the activities of macrophages at both early and late stages oflesion formation. Foam cells, a specialized form of lipid ladenmacrophage found in such lesions, are thought to be a particularlyimportant element of this process. As outlined in Example 20, engagementof ICAM-R on monocytes in the presence of oxidated phospholipid elicitssecretion of the chemokine, MCP-1, which is potentiallypro-atherosclerotic. Therefore, it is anticipated that products of theinvention which modulate ICAM-R function could be utilized to block thisprocess.

As outlined in Example 18, ICAM-R expression on vascular endothelialcells occurs selectively on neovascularizing sites found in solid tumorsand benign angiomas. Therefore, it is anticipated that products of theinvention such as monoclonal antibodies specific to ICAM-R may be usedtherapeutically either on their own or when conjugated to other moieties(e.g., toxins, radionuclides) to therapeutically target and/or detectthe presence of such neovascularizing sites.

EXAMPLE 30

Experiments were performed in which treatment with ICAM-R-specificmonoclonal antibody ICR-8.1 prevented development of graft-versus-hostdisease (GVHD) in severe combined immune deficient (SCID) mice engraftedwith human T cells.

SCID mice produce few functional lymphocytes and therefore are incapableof rejecting human peripheral blood mononuclear cell (Hu-PBMC) grafts.After transplant into SCID hosts, human T-cells present in Hu-PBMCgrafts mount an immune response directed against recipienthistocompatibility antigens resulting in GVHD. The symptoms of GVHD inHu-PBMC/SCID chimeras (e.g., skin, gut, liver and lung pathology; weightloss; and hair loss) closely resemble symptoms of severe clinical GVHDwhich occur in many leukemia patients following bone marrowtransplantation.

In clinical bone marrow transplantation, the transfer of donor bonemarrow cells is preceded by ablative therapy to eliminate therecipient's lymphoid system and residual malignant cells. Repopulationof recipient lymphoid organs with donor-derived lymphocytes is essentialfor recovery of immune function. Thus, although repopulation of hostlymphoid tissue (e.g., spleen) with donor lymphocytes is necessary forgraft function, histocompatibility antigen-driven GVH reactions in otherrecipient organs (e.g., gut) infiltrated by donor T cells result inGVHD.

Because lymphoid repopulation and antigen-driven T cell infiltrationinto target organs are thought to involve distinct mechanisms andbecause ICR-8.1 antibody blocks histocompatibility antigen-driven T-cellactivation, the ability of ICR-8.1 antibody to inhibit lethal GVHD inHu-PBMC/SCID chimeras was tested. Female CB-17 scid/scid mice werepurchased from Charles River Laboratories (Wilmington, Mass.) and housedin sterile microisolator cages with sterile bedding, food, and water.All procedures that require handling of the immune compromised animalswere performed in a sterile biosafety cabinet using aseptic technique.The mice were allowed to acclimate in the facility for seven days priorto transplant of Hu-PBMC. One day prior to transplant, each mouse wasear-tagged for identification, weighed, and injected with 0.02 ml ofanti-Asialo GM-1 (WAKO Chemicals, Richmond, Va.). See Sandhu et al., J.Immunol., 152: 3806-3813 (1994).

Hu-PBMC were prepared on the day of transplant as follows. Freshlydonated human peripheral blood from one healthy volunteer was layeredonto Histopaque gradients (Sigma Chemical Co., St. Louis, Mo.) andcentrifuged according to the manufacturer's specifications (500×g, 18°C. for 25 minutes without brake). Hu-PBMC were harvested from eachHistopaque gradient and washed three times (300×g, 18° C. for 8 minutes)in phosphate buffered saline (PBS). Trypan Blue (Life Technologies,Grand Island, N.Y.) excluding cells were counted using a hemacytometerand suspended at a concentration of 0.6-1.0×10⁸ cells/ml PBS.Anti-Asialo GM-1 treated mice were then placed in a sterile rig andexposed to 304 Gy of total body irradiation emitted from a ¹³⁷ Cs source(0.6 Gy/minute). Hu-PBMC were transplanted into each animal by injecting0.5 ml of the Hu-PBMC cell suspension intraperitoneally.

Engraftment of Hu-PBMC was assessed ten days after transplant asfollows. A small amount of blood (about 0.1 ml) was collected from theretro-orbital sinus of each recipient mouse and placed into a solutioncontaining 0.5 ml PBS+20 USP Units Sodium Heparin (Elkins-Sinn, Inc.,Cherry Hill, N.J.). The erythrocytes in each sample were lysed by tworounds of incubation in 4 ml NH₄ Cl buffer (150 mM, pH 7.4, 18° C. forthree minutes), each followed by quenching with 10 ml PBS andcentrifugation (300×g, 18° C. for 8 minutes). To detect the presence ofhuman T cells, each leukocyte pellet was incubated with a cocktailcontaining the fluorochrome-conjugated antibodies anti-HLA-A, B,C-Phycoerythrin conjugate (Pharmigen, San Diego, Calif.) andanti-CD3-Fluorescein Isothiocyanate conjugate (Pharmigen) in PBS+1%bovine serum albumin+0.1% NaN₃ for 30 minutes on ice. Engraftment,measured as the perentage of CD3⁺, HLA-A,B,C⁺ cells, was determinedusing flow cytometric analysis (FACSCAN, Becton DickinsonImmunocytometry, San Jose, Calif.).

Each recipient mouse was assigned to either PBS or ICR-8.1 treatmentgroups based on the respective degree of its engraftment (percentage ofhuman cells in peripheral blood leukocyte population) as follows. Afterranking the recipients in order of engraftment, the recipient with thehighest percentage of engraftment was assigned to either the PBS orICR-8.1 treatment group based on a coin flip. Each successive recipientthen received alternating group assignments. Beginning eleven days aftertransplant of Hu-PBMC, recipients were treated with PBS or ICR-8.1 (5mg/mouse/i.v./three times weekly). Recipient weights were recorded twiceeach week and survival was monitored daily. Spleen and gut (stomach)tissue was collected from dead recipients and frozen at -70° C. inO.C.T. Compound (Miles, Inc., Elkhart, Ind.). Sections were stained withbiotinylated anti-HLA-A,B,C, anti-CD3, or negative control antibodiesusing standard immunohistochemical techniques.

Seven of eleven (64%) PBS treated chimeras died within fifty days aftertransplant and all showed severe infiltration of human T-cells into gutepithelium as well as dramatic repopulation of spleen with humanlymphoid cells. In contrast, none of the animals treated with ICR-8.1(0/12) developed lethal GVHD reactions within the same time period. Inaddition, ICR-8.1 treatment reduced human T-cell infiltration withoutpreventing splenic repopulation.

The foregoing results indicate that ICR-8.1 treatment inhibits humanT-cell mediated lethal GVHD by blocking histocompatibilityantigen-driven T-cell activation without affecting engraftment of humanlymphoid cells in host lymphoid tissue.

EXAMPLE 31

Canine and rabbit ICAM-R polynucleotide sequences were isolated for use,for example, in generating ICAM-R reagents useful in canine and rabbitanimal models of disease states.

A. Isolation of Canine Polynucleotide Sequences

A canine spleen PBMC cDNA library (Stratagene) was probed with a humanICAM-R Pst I fragment corresponding to most of the first four domains ofICAM-R (nucleotides 171 to 1137 of SEQ ID NO: 2). The library was platedand transferred to nylon membranes as described in Example 3. The gelpurified human ICAM-R PstI fragment was radiolabeled using theBoehringer Mannheim random prime Kit. The filters were hybridized at 42°C. overnight in a solution of 40% formamide, 5× SSPE, 5× Denhardts, 50μg/ml denatured salmon sperm and 0.1% SDS with 10⁶ dpM/ml probe. Thefilters were washed in a solution of 2× SSPE and 0.1% SDS at roomtemperature then exposed to X-ray film overnight. Positive clones wereidentified with a subsequent round of screening. The phagemids werereleased following library manufacturer's suggested protocol. PlasmidDNA was prepared from each clone using the Wizard miniprep system(Promega, Madison, Wis.). The longest clone (#2) was sequenced in itsentirety. The DNA and deduced amino acid sequence of the 1693 bp cloneare presented in SEQ ID NOs: 118 and 119, respectively. In comparison tothe human sequence, the clone lacks approximately 54 bases at its 5' end(which encode sixteen amino acids of the leader sequence) but extendsthrough to the poly A tail. DNA alignment with human ICAM-R showed anoverall sequence identity of 76%. Alignment with a partial clone ofcanine ICAM-1 revealed that the gene cloned was not ICAM-1. Amino acidalignments with human ICAM-1 and ICAM-R revealed an overall sequenceidentity of 47% and 66% respectively.

B. Generation of Soluble Canine ICAM-R/IgG4 Chimeric Protein

A fragment of canine DNA coding for ICAM-R domains 1-5 (nucleotides 1 to1401 of SEQ ID NO: 118) was generated with appropriate restriction sitesat the ends for cloning into the pDCS1 vector containing the IgG4 Fcregion. pDCS1 is a modified pRC/CMV (Invitrogen) mammalian expressionvector. A DHFR gene has been added as well as the signal sequence frompHF2G. Canine ICAM-R domains 1-5 were cloned in frame downstream fromthe sequence followed by the IgG4 Sc region. The canine fragment wasgenerated by a two step PCR reaction using a 5' oligonucleotide thatcontained a BamHI site and a 3' oligonucleotide that contained an XhoIsite. In addition, there was one internal XhoI site at position 1120that needed to be eliminated to facilitate cloning. To do this,overlapping oligonucleotides were prepared encoding a singleconservative nucleotide change, eliminating the XhoI site withoutaffecting the amino acid sequence. Two primary PCR reactions werecarried out to generate a 5' fragment and a 3' fragment that overlappedin the region of the altered XhoI site. Both reactions were carried outin 25 μl volumes containing dNTPs (2 mM), Perkin Elmer Buffer with 2 mMMgCl₂, oligonucleotides (10 μg/ml), and Perkin Elmer AmptiTaq (1 unit).The template for the reactions was 2 ng of canine ICAM-R plasmid DNA.PCR conditions included 30 cycles of denaturation (94° C., 1 minute),annealing (55° C., 2 minutes) and extension (72° C., 4 minutes). The PCRDNA generated from each reaction was gel purified using a Qiagen gelextraction kit (Chatsworth, Calif.). These fragments were mixed andadded to a secondary PCR reaction to regenerate the full fragmentencoding domains 1-5. This reaction used 1/50th of the material isolatedin the primary reactions as template with the outer 5' and 3'oligonucleotides and the other PCR constituents described above. Theproduct of this reaction was approximately 1.45 kb and did not containan internal XhoI site. This DNA fragment was gel purified, cloned intothe pCRII vector (Invitrogen, San Diego, Calif.) and sequenced. PCRclone A-1 included the BamHI site at the 5' end, a conservativenucleotide change (T to C) at bp 486, the conservative point mutation inthe XhoI site at bp 1051, a mutation at bp 1364 resulting in an aminoacid substitution (Asn to Thr), and the XhoI site in frame at the 3'end. The amino acid substitution was located in the second to lastposition of the canine ICAM-R fragment and therefore was felt to be ofminimal consequence for the functional binding or generation ofmonoclonal antibodies. PCR clone A-1 was removed from the PCRIl vectorby digestion with BamHI and Xho I, purified and ligated into the pDCS1vector with IgGI Fc region. Plasmids were sequenced to confirm properorientation of the gene fragments and reading frame.

Canine ICAM-R/IgG4 fusion protein was generated by transfecting theexpression plasmid into Cos7 cells using the DEAE dextran method,followed by periodic harvesting of the culture media that contained thesecreted protein. The ICAM-R/IgG4 fusion protein was purified on aProcep A column (Bioprocessing Ltd, England). Briefly, a column volumeof 0.4-0.8 mls Procep A was washed with 60 column volumes (cv) of Tris35 mM, NaCl 150 mM pH 7.5. The collected supernatants were passed overthe column two times at <60 cv/hr. The column was then washed in 20 cv'sof the Tris/NaCl buffer, followed by 20 cv's of 0.55 M Diethanolamine pH8.5 and 20 cv's of 50 mM citric acid pH 5.0. The IgG4 fusion protein waseluted in 1 ml volumes of 50 mM citric acid pH 3.0 and neutralized with1/10 volume 1 M Tris pH 9.5. The protein concentration was determined byOD280. Purity was gauged by running a sample of the protein in an SDSPAGE. The fusion protein was approximately 80% pure, running atapproximately 120 KD under denaturing conditions.

C. Generation of Monoclonal Antibodies to Canine ICAM-R/IgG4 Protein

Balb/c mice were immunized subcutaneously with 50 μg canine ICAM-R/IgG4emulsified in Complete Freund's Adjuvant. Two weeks later, the mice wereboosted with the same amount of antigen emulsified in IncompleteFreund's Adjuvant. Tertiary and all subsequent boosts were givenintraperitoneally (IP) in soluble form. The immune sera from the micewas assessed for its ability to stain dog PBLs in a FACS assay as wellas bind to the ICAM-R part of the chimeric protein preferentially whenthe reactivity to the IgG4 was blocked. Based on this analysis, a mousewas chosen for the first-fusion (#155). Four days prior to the fusionthe mouse was given a final IP injection of 50 μg canine ICAM-R/IgG4.Altogether, 3 fusions were done (#155,161 and 168) to generatemonoclonal antibodies to canine ICAM-R. The procedure for the fusion andgrowth of the hybridomas was as described in Example 11.

The screening procedure for each fusion included a differential ELISA toassess binding to canine ICAM-R/IgG4 as distinct from binding to IgG4alone. The ELISA assay was performed essentially as described in Example11. The fusion wells were also assayed by FACS for their ability to binddog PBLs. Briefly, the dog red blood cells were lysed with ammoniumchloride, washed in PBS and resuspended in FACS media (PBS +2% FBS +Sodium Azide 0.01%). Cells (3×10⁵ cells in 50 μl) were added to 50 μl offusion well supernatant and incubated on ice for 60 minutes. Cells werepelleted by centrifugation and washed in FACS media three times. Cellswere then incubated 30 minutes on ice with sheep anti-mouse IgG Fc FITCconjugate (Sigma). The cells were washed again as before and resuspendedin PBS containing 1% paraformaldehyde. Samples were analyzed on theBecton Dickinson Facscan. These parameters were followed through thecloning process.

D. Characterization of Monoclonal Antibodies

1. Adhesion Assay

In addition to testing reactivity of the monoclonal antibodies to thecanine ICAM-R/IgG4 chimeric protein and to dog PBL, the antibodies werealso tested for their ability to block ICAM-R dependent adhesion. Theabsence of an appropriate dog lymphoid cell line for these assaysnecessitated use of a human B cell line (JY) to determine if human LFA-1(CD18/CD11a) could bind canine ICAM-R. In this assay, JY cells werelabeled with calcein by adding 8 μl (1 mg/ml) to 1 ml cells followed byan incubation at 37° C. for 20 minutes. The cells were washed andresuspended at 1×10⁶ cells/ml RPMI (Gibco) containing 0.2% BSA. AnImmulon4 96 well plate was coated overnight at 4° C. with 200 ng ofcanine ICAM-R/IgG4 in a 50 μl volume of coating buffer (50 mM NaHCO3 pH9.4). The coating mixture was removed and the unbound surface of theplate was blocked with 250 μl of PBS containing 1% BSA at 37° C. for 60minutes. Background binding to the blocking agent alone was assessed byblocking a series of wells that were not coated with canine ICAM-R/IgG4.Some of the wells coated with canine ICAM-R/IgG4 were pretreated withmonoclonal antibodies to ICAM-R (10 μg/ml) for 20-30 minus at roomtemperature in a volume of 100 μl. Alternatively, some of the labeled JYcells were preincubated with 10 μg/ml monoclonal antibody TS1.18.Following the preincubation of either the cells or the wells, 200 μl oflabeled cells were added to each well. The plates were counted on aMillipore Cytofluor 2300 to establish a reading for the amount oflabeled cells put into each well before washing. The cells were spundown onto the bottom of the plate at 500 rpm for 2 seconds then allowedto incubate for 40 minutes at 37° C. The wash step involved gentleinversion into a bath of PBS+0.1 % BSA (warmed to 37° C.). The platesremained inverted in the bath for 20 minutes before they were carefullyremoved. The plate was then read again on the Cytofluor to establish the% of cells remaining in each remaining in each well. Controls for eachassay include an assessment of the background binding to the blockingagent, maximal binding to canine ICAM-R, and the amount of bindingattributed to CD18 as blocked by TS1.18. Each monoclonal antibody wastested in triplicate in each assay.

From the monoclonal antibodies cloned in the first fusion (#155), 155D,155E and 155Z were effective at blocking the CD18 dependent adhesion(>50%) of JY cells to canine ICAM-R. Monoclonal antibodies 161B, 161G,161H and 161J from the second fusion were also effective blockers ofadhesion. Monoclonal antibodies 168A, 168B, 168C, 168E, 168G, 168I,168J, 168K and 168L were effective at blocking the CD18-dependentadhesion of Jy cells to canine ICAM-R.

E. Crossreactivity to Other Species

The monoclonal antibodies were also assessed for their crossreactivityprofiles on human and rabbit PBLs. The staining procedure and FACSanalysis were essentially as described for the canine PBLs in Section Dabove. Rabbit PBLs were stained with monclonal antibodies from the firsttwo fusions (#155 and 161). Monoclonal antibodies 155G, 155Q, 155S,155DD, 161A, 161C, 161E, 161H and 161K all stained the rabbit PBL tovarying degrees.

When tested on human PBL, a number of antibodies stained a small subsetof the lymphocytes weakly, but none showed a staining pattern similar toantibodies generated against the human ICAM-R possibly as a result oflow affinity interactions as a result of species differences. Antibodies155G, 155D, 161C, 161G and 161K stained weakly, while none of theantibodies from fusion 168 bound human PBL.

F. Generation of Point Mutant Canine

ICAM-R/IgG4 Chimeric Proteins

To characterize the specificity of monoclonal antibodies generated tocanine ICAM-R, a series of point mutations were made in the first andsecond domains of the ICAM-R portion of the fusion protein. Doublemutations at positions 32/33 (E32K/AS) and 37/38 (E37T/AL) in the firstdomain had previously been found to abrogate the binding of allmonoclonal antibodies that recognized the first domain of human ICAM-R.A single mutation at position 121 (L121/P) in the second domaineliminated all of the binding of those monoclonal antibodies specificfor the second domain without affecting the binding of antibodiesspecific for the first domain. Corresponding positions were chosen formutation in the canine ICAM-R/IgG4 molecule. The positions of the canineamino acids from PCR clone A-1 are displaced by one amino acid comparedto the numbering system used to describe the human sequence. Thecorresponding canine amino acid positions are given in parentheses inthe following paragraph.

Alignment of the canine ICAM-R with human ICAM-R revealed that the aminoacids at positions 32/33 were not conserved. Human ICAM-R had Glu/Lys atpositions 32/33 while canine ICAM-R had Arg/Leu (position 33/34). Thesesites were mutated to Gly/Phe using site directed mutagenesis. The aminoacids at position 37/38 (38/39) were conserved in the human and canineICAM-R sequences. These amino acids were mutated to Asp/Ser by the samemethod. The domain 2 mutant was the same as outlined in the human ICAM-Ranalysis, a leucine was changed to a proline at position 121 (position122 in canine). Clones encoding each mutant were sequenced through themutagenized area to detect those that contained the desired sequence. Asingle clone from each group was identified and removed from the pCRIIvector with BamHI and XhoI. The mutant fragments were cloned into thepCDS1 vector with the IgG4 Fc fragment and expressed in Cos7 cells.Analysis of the mutant chimeric proteins on SDS PAGE revealed a band atabout 120 KD under denaturing conditions. Overall purity was comparableto that seen with the wildtype protein (approximately 80%).

G. Domain Mapping of Antibodies to Canine ICAM-R

The canine ICAM-R/IgG4 point mutation chimeric proteins were used to mapbinding of each antibody. Reactivity to each mutant relative to thewildtype protein was assessed by ELISA. Antibodies 155D, 155E, 155Z,161A, 161C, 161G, 161H, 161I, 161J, 168A, 168C, 168E, 168G, 168H, 1681,168J, 168K and 168L appear to recognize domain 1 of canine ICAM-R, whileantibodies 155G, 161B, and 161D appear to recognize domain 2. Antibodies168B and 168F were not affected by the mutations.

H. Cloning of Rabbit Polynucleotide Sequences

A rabbit spleen cDNA library constructed in lambda UniZap (Stratagene)was probed with a radiolabeled canine PCR fragment A-1 corresponding tocanine ICAM-R domains 1-5. The library was plated and transferred tonylon membranes. The gel purified PCR fragment was radiolabeled usingthe Boehringer Mannheim random prime kit. The filters were hybridized at42° C. overnight in a solution of 40% formamide, 5× SSPE, 5× Denhardtsand 0.1% SDS with 106 dpm/ml probe. The filters were washed in asolution of 2× SSPE and 0.1% SDS at room temperature then exposed toX-ray film overnight. Positive clones were identified with a subsequentround of screening. The phagemids were released following manufacturer'ssuggested protocol (Stratagene). Plasmid DNA was prepared from eachclone using the Wizard miniprep system (Promega, Madison, Wis.). A 765bp clone (clone F) was identified in this screen and sequenced. Theclone corresponded to the 3' end of the gene, including the poly A+tail.

A new rabbit spleen cDNA library was made in lambda ZAP Express vector(Stratagene) according to manufacturer's protocol using kit reagents.Poly A+ RNA was prepared from a frozen rabbit spleen using Invitrogen'sFast Track mRNA isolation system (San Diego, Calif.). The RNA was usedto generate random primed cDNA that was adapted and cloned into thelambda ZAP express arms. The phage were packaged using Gigapack Goldpackaging extracts (Stratagene). The library was plated, transferred tonylon membranes and screened with radiolabeled partial rabbit ICAM-Rclone F. The longest clone emerging from this library was 1404 bp andencodes domain 2 through the cytoplasmic tail of rabbit ICAM-R. The DNAand deduced amino acid sequences of the clone are set out in SEQ ID NOs:119 and 120, respectively. Alignment with the full length human ICAM-Rsequence revealed 76% identity at the nucleotide level and 65% identityat the amino acid level.

The foregoing illustrative examples relate to presently preferredembodiments of the invention and numerous modifications and variationsthereof will be expected to occur to those skilled in the art. Thus onlysuch limitations as appear in the appended claims should be placed uponthe scope of the present invention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 120                                                (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 547 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 30..547                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       MetAlaThrMetValProSerValLeuTrpProArgAlaCysTrpThr                              25-20- 15                                                                     LeuLeuValCysCysLeuLeuThrProGlyValGlnGlyGlnGluPhe                              10-51                                                                         LeuLeuArgValGluProGlnAsnProValLeuSerAlaGlyGlySer                              51015                                                                         LeuPheValAsnCysSerThrAspCysProSerSerGluLysIleAla                              20253035                                                                      LeuGluThrSerLeuSerLysGluLeuValAlaSerGlyMetGlyTrp                              404550                                                                        AlaAlaPheAsnLeuSerAsnValThrGlyAsnSerArgIleLeuCys                              556065                                                                        SerValTyrCysAsnGlySerGlnIleThrGlySerSerAsnIleThr                              707580                                                                        ValTyrGlyLeuProGluArgValGluLeuAlaProLeuProProTrp                              859095                                                                        GlnProValGlyGlnAsnPheThrLeuArgCysGlnValGluGlyGly                              100105110115                                                                  SerProArgThrSerLeuThrValValLeuLeuArgTrpGluGluGlu                              120125130                                                                     LeuSerArgGlnProAlaValGluGluProAlaGluValThrAlaThr                              135140145                                                                     ValLeuAlaSerArgAspAspHisGlyAlaProPheSerCysArgThr                              150155160                                                                     GluLeuAspMetGlnProGlnGlyLeuGlyLeuPheValAsnThrSer                              165170175                                                                     AlaProArgGlnLeuArgThrPheValLeuProValThrProProArg                              180185190195                                                                  LeuValAlaProArgPheLeuGluValGluThrSerTrpProValAsp                              200205210                                                                     CysThrLeuAspGlyLeuPheProAlaSerGluAlaGlnValTyrLeu                              215220225                                                                     AlaLeuGlyAspGlnMetLeuAsnAlaThrValMetAsnHisGlyAsp                              230235240                                                                     ThrLeuThrAlaThrAlaThrAlaThrAlaArgAlaAspGlnGluGly                              245250255                                                                     AlaArgGluIleValCysAsnValThrLeuGlyGlyGluArgArgGlu                              260265270275                                                                  AlaArgGluAsnLeuThrValPheSerPheLeuGlyProIleValAsn                              280285290                                                                     LeuSerGluProThrAlaHisGluGlySerThrValThrValSerCys                              295300305                                                                     MetAlaGlyAlaArgValGlnValThrLeuAspGlyValProAlaAla                              310315320                                                                     AlaProGlyGlnThrAlaGlnLeuGlnLeuAsnAlaThrGluSerAsp                              325330335                                                                     AspGlyArgSerPhePheCysSerAlaThrLeuGluValAspGlyGlu                              340345350355                                                                  PheLeuHisArgAsnSerSerValGlnLeuArgValLeuTyrGlyPro                              360365370                                                                     LysIleAspArgAlaThrCysProGlnHisLeuLysTrpLysAspLys                              375380385                                                                     ThrArgHisValLeuGlnCysGlnAlaArgGlyAsnProTyrProGlu                              390395400                                                                     LeuArgCysLeuLysGluGlySerSerArgGluValProValGlyIle                              405410415                                                                     ProPhePheValAsnValThrHisAsnGlyThrTyrGlnCysGlnAla                              420425430435                                                                  SerSerSerArgGlyLysTyrThrLeuValValValMetAspIleGlu                              440445450                                                                     AlaPheSerSerHisPheValProValPheValAlaValLeuLeuThr                              455460465                                                                     LeuGlyValValThrIleValLeuAlaLeuMetTyrValPheArgGlu                              470475480                                                                     HisGlnArgSerGlySerTyrHisValArgGluGluSerThrTyrLeu                              485490495                                                                     ProLeuThrSerMetGlnProThrGluAlaMetGlyGluGluProSer                              500505510515                                                                  ArgAlaGlu                                                                     (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1781 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CAGCTCTCTGTCAGAATGGCCACCATGGTACCATCCGTGTTGTGGCCCAGGGCCTGCTGG60                ACTCTGCTGGTCTGCTGTCTGCTGACCCCAGGTGTCCAGGGGCAGGAGTTCCTTTTGCGG120               GTGGAGCCCCAGAACCCTGTGCTCTCTGCTGGAGGGTCCCTGTTTGTGAACTGCAGTACT180               GATTGTCCCAGCTCTGAGAAAATCGCCTTGGAGACGTCCCTATCAAAGGAGCTGGTGGCC240               AGTGGCATGGGCTGGGCAGCCTTCAATCTCAGCAACGTGACTGGCAACAGTCGGATCCTC300               TGCTCAGTGTACTGCAATGGCTCCCAGATAACAGGCTCCTCTAACATCACCGTGTACGGG360               CTCCCGGAGCGTGTGGAGCTGGCACCCCTGCCTCCTTGGCAGCCGGTGGGCCAGAACTTC420               ACCCTGCGCTGCCAAGTGGAGGGTGGGTCGCCCCGGACCAGCCTCACGGTGGTGCTGCTT480               CGCTGGGAGGAGGAGCTGAGCCGGCAGCCCGCAGTGGAGGAGCCAGCGGAGGTCACTGCC540               ACTGTGCTGGCCAGCAGAGACGACCACGGAGCCCCTTTCTCATGCCGCACAGAACTGGAC600               ATGCAGCCCCAGGGGCTGGGACTGTTCGTGAACACCTCAGCCCCCCGCCAGCTCCGAACC660               TTTGTCCTGCCCGTGACCCCCCCGCGCCTCGTGGCCCCCCGGTTCTTGGAGGTGGAAACG720               TCGTGGCCGGTGGACTGCACCCTAGACGGGCTTTTTCCAGCCTCAGAGGCCCAGGTCTAC780               CTGGCGCTGGGGGACCAGATGCTGAATGCGACAGTCATGAACCACGGGGACACGCTAACG840               GCCACAGCCACAGCCACGGCGCGCGCGGATCAGGAGGGTGCCCGGGAGATCGTCTGCAAC900               GTGACCCTAGGGGGCGAGAGACGGGAGGCCCGGGAGAACTTGACGGTCTTTAGCTTCCTA960               GGACCCATTGTGAACCTCAGCGAGCCCACCGCCCATGAGGGGTCCACAGTGACCGTGAGT1020              TGCATGGCTGGGGCTCGAGTCCAGGTCACGCTGGACGGAGTTCCGGCCGCGGCCCCGGGG1080              CAGACAGCTCAACTTCAGCTAAATGCTACCGAGAGTGACGACGGACGCAGCTTCTTCTGC1140              AGTGCCACTCTCGAGGTGGACGGCGAGTTCTTGCACAGGAACAGTAGCGTCCAGCTGCGA1200              GTCCTGTATGGTCCCAAAATTGACCGAGCCACATGCCCCCAGCACTTGAAATGGAAAGAT1260              AAAACGAGACACGTCCTGCAGTGCCAAGCCAGGGGCAACCCGTACCCCGAGCTGCGGTGT1320              TTGAAGGAAGGCTCCAGCCGGGAGGTGCCGGTGGGGATCCCGTTCTTCGTCAACGTAACA1380              CATAATGGTACTTATCAGTGCCAAGCGTCCAGCTCACGAGGCAAATACACCCTGGTCGTG1440              GTGATGGACATTGAGGCTGGGAGCTCCCACTTTGTCCCCGTCTTCGTGGCGGTGTTACTG1500              ACCCTGGGCGTGGTGACTATCGTACTGGCCTTAATGTACGTCTTCAGGGAGCACCAACGG1560              AGCGGCAGTTACCATGTTAGGGAGGAGAGCACCTATCTGCCCCTCACGTCTATGCAGCCG1620              ACAGAAGCAATGGGGGAAGAACCGTCCAGAGCTGAGTGACGCTGGGATCCGGGATCAAAG1680              TTGGCGGGGGCTTGGCTGTGCCCTCAGATTCCGCACCAATAAAGCCTTCAAACTCCCAAA1740              AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA1781                                 (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /note= "The amino acid at this                         position can be a valine, a leucine or an                                     isoleucine."                                                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "The amino acid at this                         position can be a valine, a leucine or an                                     isoleucine."                                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GlyXaaXaaXaaXaaXaaXaaCys                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "The amino acid at this                         position can be a valine or an alanine."                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AspXaaGlyXaaTyrXaaCysXaaXaa                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /note= "The amino acid at this                         position can be an asparagine or a serine."                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /note= "The amino acid at this                         position can be a lysine or a phenylalanine."                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "The amino acid at this                         position can be an lysine or an isoleucine."                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "The amino acid at this                         position can be an arginine or a glutamic acid."                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GlyLysXaaXaaThrXaaXaaCys                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "The amino acid at this                         position can be a aspartic acid or a glutamic                                 acid."                                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "The amino acid at this                         position can be a histidine or an aspartic acid."                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /note= "The amino acid at this                         position can be a histidine or a glycine."                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /note= "The amino acid at this                         position can be a glycine or a histidine."                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note= "The amino acid at this                         position can be an alanine or an arginine."                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       XaaXaaXaaXaaXaaAsnPheSerCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ATTCTGCAGGCAARAAYCTSACHMTBMGSTG31                                             (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ATTCTGCAGGCAARAGYTTYACHMTBGARTG31                                             (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       ATTCTGCAGGCAARTCYTTYACHMTBGARTG31                                             (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      ATTTCTAGARAARTTRGCSCCRTGRTSRTC30                                              (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      ATTTCTAGARAARTTSCKRTGSCCRTSKTC30                                              (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GAGACTCTGCACTATGAGACCTTCG25                                                   (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CAGGTGATTCTCATGCAGAGTCCAGG26                                                  (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      CCGACATGCTGGTAAGTGTGTCCAA25                                                   (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GACCATGAGGTGCCAAG17                                                           (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      ATGGTCGTCTCTGCTGG17                                                           (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      TTCACCCTGCGCTGCCAA18                                                          (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      AAAGGGGCTCCGTGGTCG18                                                          (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      CCGGTTCTTGGAGGTGGAA19                                                         (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      CATGACTGTCGCATTCAGCA20                                                        (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      GCAAGAACCTTACCCTAC18                                                          (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      GAAATTGGCTCCATGGTGA19                                                         (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      CCGGGTCCTAGAGGTGGACACGCA24                                                    (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      TGCAGTGTCTCCTGGCTCTGGTTC24                                                    (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      AspGlyGlnSerThr                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      GlyAspGlnArgLeu                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      CTGCCCCTGAATCACCCTCGA21                                                       (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      GTAAAACGACGGCCAGT17                                                           (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      CAGGTCCCGGTCATCATCATCATCATCATTAAT33                                           (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      TAGATTAATGATGATGATGATGATGACCGGGACCTGAGCT40                                    (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      GGAAGCTTAGACAGATGGGGGTGTCGTTTTG31                                             (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      GCTATCGGATCCACTGGATGGTGGGAAGATGGA33                                           (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      CGATACGAATTCSADGTRCAGCTKMAGGAGTCRGGA36                                        (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      CGATACGAATTCSAGGTYCARCTKCARCARYCTGG35                                         (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      CGATACGAATTCGARGTGAAGCTKSWSGAGWCTGG35                                         (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      CGATACGAATTCAGGTSMARCTGCAGSAGTCWG33                                           (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      CGATACGAATTCSAGGTSMARCTGCAGSARHC32                                            (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      CGATACGAATTCSAAAWTGTKCTSACCCAGTCTCCA36                                        (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      CGATACGAATTCGACATTGTGMTGWCMCARTCTCC35                                         (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      CGATACGAATTCGATRTTKTGATGACYCARRCTSCA36                                        (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      CGATACGAATTCGAYATYSWGATGACMCAGWCTMC35                                         (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 418 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      GAATTCATGGRATGGAGCTGGRTCWTBHTCTTCCTGTCAGGGGCTGCAGGTGCCCACTCT60                GAGATCCAGCTGCAGCAGACTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATA120               TCTTGCAAGGCTTCTGGTTATTCATTCACTGACTGCATCATACTCTGGGTGAAGCAGAGC180               CATGGAAAGGGCCTTGAGTGGATTGGAAAAATTAATCCTTACTTTGGTACTACTACCTAT240               AATCTGAAATTCAAGGGCAAGGCCACATTGACTGTAGACAAATCTTCCAGCACAGCCCAC300               ATGCAGCTCAACAGTCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAAAGGAG360               GCCTACCCAGATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAG418                 (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 137 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      MetXaaTrpSerTrpXaaXaaXaaPheLeuSerGlyAlaAlaGlyAla                              151015                                                                        HisSerGluIleGlnLeuGlnGlnThrGlyProGluLeuValLysPro                              202530                                                                        GlyAlaSerValLysIleSerCysLysAlaSerGlyTyrSerPheThr                              354045                                                                        AspCysIleIleLeuTrpValLysGlnSerHisGlyLysGlyLeuGlu                              505560                                                                        TrpIleGlyLysIleAsnProTyrPheGlyThrThrThrTyrAsnLeu                              65707580                                                                      LysPheLysGlyLysAlaThrLeuThrValAspLysSerSerSerThr                              859095                                                                        AlaHisMetGlnLeuAsnSerLeuThrSerGluAspSerAlaValTyr                              100105110                                                                     TyrCysAlaArgLysGluAlaTyrProAspAlaMetAspTyrTrpGly                              115120125                                                                     GlnGlyThrSerValThrValSerSer                                                   130135                                                                        (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 384 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      GTCGACAGGCTGTTGGTGCTGATGYTCTGGATTCCTGTTTCCAGTAGTGACGCTGTGATG60                ACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGA120               TCTAGTCAGAGCCTTGTACACAGTAATGGAGACACCTATTTACATTGGTACCTGCAGAAG180               CCAGGCCAGTCTCCACAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCA240               GACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGCTCAGCAGAGTGGAG300               GCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGTACACGTTCGGA360               GGGGGGACCAAGCTGGAAATAAAA384                                                   (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 127 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      ArgLeuLeuValLeuMetXaaTrpIleProValSerSerSerAspAla                              151015                                                                        ValMetThrGlnThrProLeuSerLeuProValSerLeuGlyAspGln                              202530                                                                        AlaSerIleSerCysArgSerSerGlnSerLeuValHisSerAsnGly                              354045                                                                        AspThrTyrLeuHisTrpTyrLeuGlnLysProGlyGlnSerProGln                              505560                                                                        LeuLeuIleTyrLysValSerAsnArgPheSerGlyValProAspArg                              65707580                                                                      PheGlySerGlySerGlySerGlyThrAspPheThrLeuLysLeuSer                              859095                                                                        ArgValGluAlaGluAspLeuGlyValTyrPheCysSerGlnSerThr                              100105110                                                                     HisValProTyrThrPheGlyGlyGlyThrLysLeuGluIleLys                                 115120125                                                                     (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      GGCCTGTCGCACCCAGAGTATGATGCAGTCAGTGAAGRTGTATCC45                               (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 90 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      TGTGTCCRCGGTAATGGTCACTCTGCCCTTGAATTTCAGATTATAGGTAGTAGTACCAAA60                GTAAGGATTAATTTTTCCCATCCATTCGAG90                                              (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 63 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      TCCTTGGCCCCCAGTAGTCCATAGCATCTGGGTAGGCCTCCTTTCTTGCACAGTAATACA60                CGG63                                                                         (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      GTAAAACGACGGCCAGT17                                                           (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      AACAGCTATGACCATG16                                                            (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      ACCATTACCGCGGACACATCCAC23                                                     (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 395 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      GCGGCCGCAGGTGTCCAGTCCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAG60                CCTGGGGCTAGCGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACTGACTGCATC120               ATACTCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAATGGATGGGAAAAATTAATCCT180               TACTTTGGTACTACTACCTATAATCTGAAATTCAAGGGCAGAGTGACCATTACCGCGGAC240               ACATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTG300               TATTACTGTGCAAGAAAGGAGGCCTACCCAGATGCTATGGACTACTGGGGCCAAGGAACC360               CTGGTCACCGTCTCCTCAGGTGAGTCCTTGGATCC395                                        (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 123 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      GlyValGlnSerGlnValGlnLeuValGlnSerGlyAlaGluValLys                              151015                                                                        LysProGlyAlaSerValLysValSerCysLysAlaSerGlyTyrThr                              202530                                                                        PheThrAspCysIleIleLeuTrpValArgGlnAlaProGlyGlnGly                              354045                                                                        LeuGluTrpMetGlyLysIleAsnProTyrPheGlyThrThrThrTyr                              505560                                                                        AsnLeuLysPheLysGlyArgValThrIleThrAlaAspThrSerThr                              65707580                                                                      SerThrAlaTyrMetGluLeuSerSerLeuArgSerGluAspThrAla                              859095                                                                        ValTyrTyrCysAlaArgLysGluAlaTyrProAspAlaMetAspTyr                              100105110                                                                     TrpGlyGlnGlyThrLeuValThrValSerSer                                             115120                                                                        (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      TACTACCTATGCTCAGAAATTCCAGGGCAGAG32                                            (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      CTCTGCCCTGGAATTTCTGAGCATAGGTAGTAG33                                           (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      CACAGGTGTCCACTCCCAGATCCAGCTGG29                                               (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      TGGGAGTGGACACCTGTGGAGAGAAAGGCAAAGTGG36                                        (2) INFORMATION FOR SEQ ID NO:58:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                      CACAGGTGTCCACTCCCAGATCCAGCTGGTGCAGACTGGGGC42                                  (2) INFORMATION FOR SEQ ID NO:59:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                      GCTCTCCAGGAGTGACAGGCAGGG24                                                    (2) INFORMATION FOR SEQ ID NO:60:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 52 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                      TTGCGGCCGCAGGTGTCCAGTCCGACATTGTAATGACCCAGTCTCCACTCTC52                        (2) INFORMATION FOR SEQ ID NO:61:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                      TCACTCCTGGAGAGCCAGCCTCCATCTCTTGCAGA35                                         (2) INFORMATION FOR SEQ ID NO:62:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                      CCTCAGCCTCCACTCTGCTGATCTTGAGTGT31                                             (2) INFORMATION FOR SEQ ID NO:63:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                      AGAGTGGAGGCTGAGGATGTGGGAGTTTATTACTGCTCTC40                                    (2) INFORMATION FOR SEQ ID NO:64:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                      TTGGATCCTAAGTACTTACGTTTTATTTCCACCTTGGTCCCCTGTCCG48                            (2) INFORMATION FOR SEQ ID NO:65:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 375 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                      GCGGCCGCAGGTGTCCAGTCCGACATTGTAATGACCCAGTCTCCACTCTCCCTGCCTGTC60                ACTCCTGGAGAGCCAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAAT120               GGAGACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCACAGCTCCTGATC180               TACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGG240               ACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATGTGGGAGTTTATTACTGC300               TCTCAAAGTACACATGTTCCGTACACGTTCGGACAGGGGACCAAGGTGGAAATAAAACGT360               AAGTACTTAGGATCC375                                                            (2) INFORMATION FOR SEQ ID NO:66:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 116 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                      GlyValGlnSerAspIleValMetThrGlnSerProLeuSerLeuPro                              151015                                                                        ValThrProGlyGluProAlaSerIleSerCysArgSerSerGlnSer                              202530                                                                        LeuValHisSerAsnGlyAspThrTyrLeuHisTrpTyrLeuGlnLys                              354045                                                                        ProGlyGlnSerProGlnLeuLeuIleTyrLysValSerAsnArgPhe                              505560                                                                        SerGlyValProAspArgPheSerGlySerGlySerGlyThrAspPhe                              65707580                                                                      ThrLeuLysIleSerArgValGluAlaGluAspValGlyValTyrTyr                              859095                                                                        CysSerGlnSerThrHisValProTyrThrPheGlyGlnGlyThrLys                              100105110                                                                     ValGluIleLys                                                                  115                                                                           (2) INFORMATION FOR SEQ ID NO:67:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                      TTGCGGCCGCAGGTGTCCAGTCCGAGGTGCAACTGCAGCAGTCTGGAC48                            (2) INFORMATION FOR SEQ ID NO:68:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                                      TGGATCCAAGGACTCACCTGAGGAGACGGTGACTGAGGTTCC42                                  (2) INFORMATION FOR SEQ ID NO:69:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                                      TTCTGGTTATACTTTCACTGTACT24                                                    (2) INFORMATION FOR SEQ ID NO:70:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                                      AGTCAGTGAAAGTATAACCAGAA23                                                     (2) INFORMATION FOR SEQ ID NO:71:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                                      CTCCGAGATCCAGCTGCAGCAGACTGGACC30                                              (2) INFORMATION FOR SEQ ID NO:72:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 49 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                                      CAGCTGGATCTCGGAGTGGACACCTGTGGAGAGAAAGGCAAAGTGGATG49                           (2) INFORMATION FOR SEQ ID NO:73:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                                      TTGCGGCCCGCAGGTGTCCAGTCCGACGCTGTGACCCAAAC41                                   (2) INFORMATION FOR SEQ ID NO:74:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                                      TTGGATCCTAAGTACTTACGTTTTATTTCCAGCTTGGT38                                      (2) INFORMATION FOR SEQ ID NO:75:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                                      GCTATCGGATCCGGARCCAGTTGTAYCTCCACACAC36                                        (2) INFORMATION FOR SEQ ID NO:76:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:                                      CGATACGAATTCSAGGTSMARCTGCAGSAGTCT33                                           (2) INFORMATION FOR SEQ ID NO:77:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 422 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:                                      TGGAATTCATGGRATGGAGCTGGRTCWTBHTCTTCCTGTTTTCAGTAACTGCAGGTGTCC60                ACTCCCAGGTCCAGCTTCAGCAGTCTGGGGCTGAACTGGCAAAACCTGGGGCCTCAGTGA120               AGATGTCCTGCAAGGCTTCTGGCTACACCTTTACTGTTTACTGGATGCACTGGGTAAAAC180               AGAGGCCTGGACAGGGTCTAGAATGGATTGGATACATTAATCCTAACACTGATTATACTG240               AGTACAATCAGAGGTTCCAGGACAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAG300               CCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAT360               GGGGGGGTAACTCCTATGGTTTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCT420               CA422                                                                         (2) INFORMATION FOR SEQ ID NO:78:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 138 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:                                      MetXaaTrpSerTrpXaaXaaXaaPheLeuPheSerValThrAlaGly                              151015                                                                        ValHisSerGlnValGlnLeuGlnGlnSerGlyAlaGluLeuAlaAsp                              202530                                                                        ProGlyAlaSerValLysMetSerCysLysAlaSerGlyTyrThrPhe                              354045                                                                        ThrValTyrTrpMetHisTrpValLysGlnArgProGlyGlnGlyLeu                              505560                                                                        GluTrpIleGlyTyrIleAsnProAsnThrAspTyrThrGluTyrAsn                              65707580                                                                      GlnArgPheGlnAspLysAlaThrLeuThrAlaAspLysSerSerSer                              859095                                                                        ThrAlaTyrMetGlnLeuSerSerLeuThrSerGluAspSerAlaVal                              100105110                                                                     TyrTyrCysAlaArgTrpGlyGlyAsnSerTyrGlyLeuAspTyrTrp                              115120125                                                                     GlyGlnGlyThrSerValThrValSerSer                                                130135                                                                        (2) INFORMATION FOR SEQ ID NO:79:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 390 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:                                      AATGTCGACATGGATTTTCAAGTGATTTTCAGCTTCCTGCTAATGAGTGCCTCAGTCATT60                ATGTCCAGGGGACAAATTGTTCTCACCCAGTCTCCAGCACTCATGTCTGCATCTCCAGGG120               GAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATTTATTGGTACCAG180               CAGAAGCCAAGATCCTCCCCCAAACCCTGGATTTATCTCACATCCAACCTGGCTTCTGGA240               GTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGGCCTCTTACTCTCTCACAATCAGCATC300               ATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAAGAGTATCCCACTCACG360               TTCGGTGCTGGGACCAAGCTGGAGCTGAAA390                                             (2) INFORMATION FOR SEQ ID NO:80:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 127 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:                                      MetAspPheGlnValIlePheSerPheLeuLeuMetSerAlaSerVal                              151015                                                                        IleMetSerArgGlyGlnIleValLeuThrGlnSerProAlaLeuMet                              202530                                                                        SerAlaSerProGlyGluLysValThrMetThrCysSerAlaSerSer                              354045                                                                        SerValSerTyrIleTyrTrpTyrGlnGlnLysProArgSerSerPro                              505560                                                                        LysProTrpIleTyrLeuThrSerAsnLeuAlaSerGlyValProAla                              65707580                                                                      ArgPheSerGlySerGlySerGlyAlaSerTyrSerLeuThrIleSer                              859095                                                                        IleMetGluAlaGluAspAlaAlaThrTyrTyrCysGlnGlnTrpLys                              100105110                                                                     SerIleProLeuThrPheGlyAlaGlyThrLysLeuGluLeuLys                                 115120125                                                                     (2) INFORMATION FOR SEQ ID NO:81:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:                                      CCTGTCGCACCCAGTGCATCCAGTAAACAGTGAAGGTGTATCC43                                 (2) INFORMATION FOR SEQ ID NO:82:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 90 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:                                      GTCCGCGGTAATGGTCACTCTGTCCTGGAACCTCTGATTGTACTCAGTATAATCAGTGTT60                AGGATTAATGTATCCMATCCACTCGAGCCC90                                              (2) INFORMATION FOR SEQ ID NO:83:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:                                      GGCCCCAGTAGTCCAAACCATAGGAGTTACCCCCCCATCTGGCACAGTAATACACGG57                   (2) INFORMATION FOR SEQ ID NO:84:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:                                      CTCGAGTGGATGGGATACATTAA23                                                     (2) INFORMATION FOR SEQ ID NO:85:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 395 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:                                      GCGGCCGCAGGTGTCCAGTCCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAG60                CCTGGGGCTAGCGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACTGTTTACTGG120               ATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGATACATTAATCCT180               AACACTGATTATACTGAGTACAATCAGAGGTTCCAGGACAGAGTGACCATTACCGCGGAC240               ACATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTG300               TATTACTGTGCAAGATGGGGGGGTAACTCCTATGGTTTGGACTACTGGGGCCAAGGAACC360               CTGGTCACCGTGTCCTCAGGTGAGTCCTTGGATCC395                                        (2) INFORMATION FOR SEQ ID NO:86:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 123 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:                                      GlyValGlnSerGlnValGlnLeuValGlnSerGlyAlaGluValLys                              151015                                                                        LysProGlyAlaSerValLysValSerCysLysAlaSerGlyTyrThr                              202530                                                                        PheThrValTyrTrpMetHisTrpValArgGlnAlaProGlyGlnGly                              354045                                                                        LeuGluTrpMetGlyTyrIleAsnProAsnThrAspTyrThrGluTyr                              505560                                                                        AsnGlnArgPheGlnLysArgValThrIleThrAlaAspThrSerThr                              65707580                                                                      SerThrAlaTyrMetGluLeuSerSerLeuArgSerGluAspThrAla                              859095                                                                        ValTyrTyrCysAlaArgTrpGlyGlyAsnSerTyrGlyLeuAspTyr                              100105110                                                                     TrpGlyGlnGlyThrLeuValThrValSerSer                                             115120                                                                        (2) INFORMATION FOR SEQ ID NO:87:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:                                      GTGGATCCAAGGACTCACCTGAGGAG26                                                  (2) INFORMATION FOR SEQ ID NO:88:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:                                      ACCGCGGACAAATCCACGAG20                                                        (2) INFORMATION FOR SEQ ID NO:89:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:                                      CTCGTGGATTTGTCCGCGGT20                                                        (2) INFORMATION FOR SEQ ID NO:90:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:                                      CACAGGTGTGTCCACTCCCAAGTCCAGC28                                                (2) INFORMATION FOR SEQ ID NO:91:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 59 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:                                      TTCTGTTGGTACCAGTAAATGTAACTTACACTTGAGCTGGCACTGCAAGTGATGGTGAC59                 (2) INFORMATION FOR SEQ ID NO:92:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:                                      TTGATGGGACCCCAGAAGCCAGGTTGGATGTAAGATAGATCAGGAGC47                             (2) INFORMATION FOR SEQ ID NO:93:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 53 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:                                      CCCCTGGCCGAACGTGAGTGGGATACTCTTCCACTGCTGACAGTAGTAAGTTG53                       (2) INFORMATION FOR SEQ ID NO:94:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:                                      GTGAGAGTGTAGTCTGTCC19                                                         (2) INFORMATION FOR SEQ ID NO:95:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 357 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:                                      GCGGCCGCAGGTGCCAGATGTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCA60                TCTGTAGGAGACAGAGTCACCATCACTTGCAGTGCCAGCTCAAGTGTAAGTTACATTTAC120               TGGTACCAACAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATCTTACATCCAACCTG180               GCTTCTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGACTTCACTCTCACC240               ATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTACTGTCAGCAGTGGAAGAGTATC300               CCACTCACGTTCGGCCAGGGGACCAAGCTGGAGATCAAACGTAAGTACTTAGGATCC357                  (2) INFORMATION FOR SEQ ID NO:96:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 110 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:                                      GlyAlaArgCysAspIleGlnMetThrGlnSerProSerSerLeuSer                              151015                                                                        AlaSerValGlyAspArgValThrIleThrCysSerAlaSerSerSer                              202530                                                                        ValSerTyrIleTyrTrpTyrGlnGlnLysProGlyLysAlaProLys                              354045                                                                        LeuLeuIleTyrLeuThrSerAsnLeuAlaSerGlyValProSerArg                              505560                                                                        PheSerGlySerGlySerGlyThrAspPheThrLeuThrIleSerSer                              65707580                                                                      LeuGlnProGluAspPheAlaThrTyrTyrCysGlnGlnTrpLysSer                              859095                                                                        IleProLeuThrPheGlyGlnGlyThrLysLeuGluIleLys                                    100105110                                                                     (2) INFORMATION FOR SEQ ID NO:97:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:                                      CACAGGTGTCCACTCCCAAATCGTGCTGACCCAGTCTCCATCCTCCC47                             (2) INFORMATION FOR SEQ ID NO:98:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:                                      TTAAAGATCTAAGTACTTACGTTTGATCTC30                                              (2) INFORMATION FOR SEQ ID NO:99:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:                                      CACAGGTGTCCACTCCCAAGTCCAGC26                                                  (2) INFORMATION FOR SEQ ID NO:100:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:                                     TTGGATCCAAGGACTCACCTGAGGAGACGGTGACTGAGGT40                                    (2) INFORMATION FOR SEQ ID NO:101:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:                                     TTGGATCCTAAGTACTTACGTTTCAGCTCCAGCTTGGTCCCAG43                                 (2) INFORMATION FOR SEQ ID NO:102:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:                                     CAGGTGTCCACTCCCAAATTGTTCTCACCCAGTCTCCAGCACTCATG47                             (2) INFORMATION FOR SEQ ID NO:103:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:                                     ValLeuSerAlaGlyGlySerLeuPheVal                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:104:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:                                     LeuSerAlaGlyGlySerLeuPheValAsn                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:105:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:                                     AGAGGGGAGGGGTGCTAGCTCCACCCGTTCTGG33                                           (2) INFORMATION FOR SEQ ID NO:106:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106:                                     GAGCGTGTGGAGCTAGCACCCCTGCCT27                                                 (2) INFORMATION FOR SEQ ID NO:107:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:107:                                     GGGGGAGTCGCTAGCAGGACAAAGGTC27                                                 (2) INFORMATION FOR SEQ ID NO:108:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:108:                                     CGAACCTTTGTCCTGCTAGCGACCCCCCCGCGCCTC36                                        (2) INFORMATION FOR SEQ ID NO:109:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:109:                                     TGAGACCTCTGGCTTCCTTAAGATCACGTTGGGCGCCGG39                                     (2) INFORMATION FOR SEQ ID NO:110:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:                                     GACCCATTGTGAACTTAAGCGAGCCCACC29                                               (2) INFORMATION FOR SEQ ID NO:111:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:111:                                     CAGTGGGATCCTGTTAATGTACGTCTTCAGGG32                                            (2) INFORMATION FOR SEQ ID NO:112:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:112:                                     TGGGAGTTTGAAGGCTTT18                                                          (2) INFORMATION FOR SEQ ID NO:113:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:113:                                     TACATGTTAGGGAGGACAGCACCTAT26                                                  (2) INFORMATION FOR SEQ ID NO:114:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:114:                                     TACCATGTTAGGGACGAGAGCACCTAT27                                                 (2) INFORMATION FOR SEQ ID NO:115:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:115:                                     TACCATGTTAGGGAGGCCAGCACCTAT27                                                 (2) INFORMATION FOR SEQ ID NO:116:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:116:                                     TACCATGTTAGGGCCGAGAGCACCTAT27                                                 (2) INFORMATION FOR SEQ ID NO:117:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1600 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:117:                                     CTTTGACCTCCCAGGCTCAAGCGATCCTCTCACCTCAGCCTTCCAAGGAGCTGGGACTAC60                AGGCACCAGGCCCATGCTACTGGTCCCTGCTGTTTACAAATACACTAGACTTGGGTTTTA120               GCTGCTCTCTCAACACCAGGTGGGAAATGGATTGTTCGGAAGGTAGAGGAATAGAGGAGG180               GACCTGCTTAGGAAGGTGGGGGTGCTCTAGATGAGAGAGGAGGTGCTGTGACCTCAAGGT240               CTGGGGAAAGTGGGTGAGTGCAGGGCCCCATCAAGGCTCTCAACAAGGCCTCTCTCTCTG300               GAGCTCAGATGTGAAGCCCAGAAGGCAGGTCAGAGGCCTGAGGTCAGAGGCAGGTCAAAG360               GTGGGGGTSCCACCTGGTCCCCTTGGCGCCTTCCCTTGGAATGCAGTGACCACCTGCCTG420               GAAGGGTGGGCAAGGGGACAACAGGATGGGGTAAAGGCCAGTTACAAGTAGAATCCTGGA480               AGGCCGGGCGCAGTGGCTCACGCCTGTAATCCCAACACTTTGGGAGGCCAAGGCGGGCAG540               CTCACTTGAGGTCAGAAGTTTGAGACCAGCTTGGCCAACATGGTGAAACCCCGTCTCTAC600               CAAAAAGTACAAAAATTAGCCGGGCGTGGTGGCACATGCCTGTAATCCCAGCTACTCGGG660               AGGCTGAGGCAGGAAAATCGTTTGAACCCGGGAGGCAGAGGCTGCAGTGAGCCGAGATCC720               CGCCACTGCACTCCAGCCTGGGTGACAGAGTGAGACTCTGTCTCAAAACAAACAAACAAA780               AACCAAGTAGAATCCTGGATAGACAGTGGCTCAGGGACTCAGCTGTCCCCAAATCACCTA840               CCTTAGCTCTTTGTGCAGGGGATGGGGGATCCCCAAGATCTTCAAAGATAGGGAGGTATC900               CTCAAGATCTTCAAAGGTGGCCAGGCGTGGTGGCTCACGCCTGTAATCGCAACACTTTGG960               GAGGTCAAGATGAGAGGAGGCCATCTTAAGGCCAGGAGTTCAAGACCAGTCGGGGCAACA1020              TAGCAAGACTCTGCTTCTGTTTCTGTTTGTGGTTTKTGKTTTTTAATGTTTCCTCGAAAT1080              CCCACCTTAACATCTACACAATGGTGCCTACAGTTTTCCCCATTAACTTCCTCTCTGGGG1140              ACAGAATATTCCCCTACCCACCTAAGTTCAGCCAGAAGGAGATGCCTCATCCCCCCCACC1200              CAGGCTTCCCAGTGCTGAGAACAAAAGAAACCAGTTTAGTTAGCATTTCCTGTGAATTCA1260              AATGGGGGCCAGCAGTGCAACTGGGACCAGCCCATTCCCTGGGGGAGACTCAGCAGTGCT1320              GGCCACTTTGCAGAGCCCCGTCTGGGTCCCCCACCCTGGCCCCCAAGGGCCACAGCATCC1380              CCCTTCCACCCACAGGCCTGGGCAAGGTGCAGTCCCCAGACTTCTGCAATCTTAACCGCT1440              GTGCTTCCGTCGGAGTGGGAGGCCCCGCCTTTTCCCCTGCCTGCCCTTCGGGCACCTCAG1500              GAAGGCACCTTCCTCTGTCAGAATGGCCACCATGGTACCATCCGTGTTGTGGCCCAGGGC1560              CTGCTGGACTCTGCTGGTCTGCTGTCTGCTGACCCCAGGT1600                                  (2) INFORMATION FOR SEQ ID NO:118:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1693 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1587                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:118:                                     CTTCTGGTCTGCTGTCTGCCGCCCTCAGGTGCCCGGGCACAGCAATAC48                            LeuLeuValCysCysLeuProProSerGlyAlaArgAlaGlnGlnTyr                              151015                                                                        CAAATGAGGCTGGAGGTCGAGAACACTCTGGTGCCTGCTGGAGGGTCC96                            GlnMetArgLeuGluValGluAsnThrLeuValProAlaGlyGlySer                              202530                                                                        TTCTTGGTAAACTGCAGTACAGACTGCCCCAATCCTCGACTCATCATT144                           PheLeuValAsnCysSerThrAspCysProAsnProArgLeuIleIle                              354045                                                                        CTAGAGACATCCCTAGCCAAGAAGCCAGTGGGCAACGGCCTGGGCTGG192                           LeuGluThrSerLeuAlaLysLysProValGlyAsnGlyLeuGlyTrp                              505560                                                                        GCAGCCTTCCTGCTAAGCAATGTGACTAGTGACAGCCAGGTCCTCTGC240                           AlaAlaPheLeuLeuSerAsnValThrSerAspSerGlnValLeuCys                              65707580                                                                      TCCGGCTTCTGCAATGACATCCAGATGGTAGGCTCCTCTGAGATCACA288                           SerGlyPheCysAsnAspIleGlnMetValGlySerSerGluIleThr                              859095                                                                        GTATACCGGTTCCCGGAGCGAGTGGAGCTGGCACCCCTACCCCGCTGG336                           ValTyrArgPheProGluArgValGluLeuAlaProLeuProArgTrp                              100105110                                                                     CAGCCCGTGGGTGAGAACCTCACCATGACCTGCCAGGTGGCAGGCGGG384                           GlnProValGlyGluAsnLeuThrMetThrCysGlnValAlaGlyGly                              115120125                                                                     GCGCCCCGGACCAACCTCACGGTGGTGCTGCTCCGCGGGGAGGAGGAG432                           AlaProArgThrAsnLeuThrValValLeuLeuArgGlyGluGluGlu                              130135140                                                                     CTGAGCCGGCAACCGGCCGTCGGGGAGCCAGCCGAGGTCACGTTCACG480                           LeuSerArgGlnProAlaValGlyGluProAlaGluValThrPheThr                              145150155160                                                                  GTGGCGGTGGGCAGGGAGGACCACCTCGCCAACTTCTCGTGTCGCACG528                           ValAlaValGlyArgGluAspHisLeuAlaAsnPheSerCysArgThr                              165170175                                                                     GACCTGGACCTGAGGCCCCGAGGGCTGGGATTATTCCAGAACAGCTCG576                           AspLeuAspLeuArgProArgGlyLeuGlyLeuPheGlnAsnSerSer                              180185190                                                                     GCGCCCAGGCAGCTCCGAACCTTTGCACTGCCCATCACGGCTCCGCAC624                           AlaProArgGlnLeuArgThrPheAlaLeuProIleThrAlaProHis                              195200205                                                                     CTCGTTGTCCCTCGGATCTTGGAGGTGGGAACGACTCGGTCGGTGAAC672                           LeuValValProArgIleLeuGluValGlyThrThrArgSerValAsn                              210215220                                                                     TGCATCCTGGAAGGATTGTTCCCGGCCTCCGAGGCCCAAGTCCACTTG720                           CysIleLeuGluGlyLeuPheProAlaSerGluAlaGlnValHisLeu                              225230235240                                                                  GCGCTGGGGAACCAGACGCTGAACTCTACAGTCGAGAGCCACGGGGAC768                           AlaLeuGlyAsnGlnThrLeuAsnSerThrValGluSerHisGlyAsp                              245250255                                                                     ACGATCAGTGCCACAGCCACAGCCGTAGCGAGAGCGGAGCAGGAGGGC816                           ThrIleSerAlaThrAlaThrAlaValAlaArgAlaGluGlnGluGly                              260265270                                                                     GCACAGGAGATAGTCTGCAACATAACGTTGGGGAACGACGGCCGGGAG864                           AlaGlnGluIleValCysAsnIleThrLeuGlyAsnAspGlyArgGlu                              275280285                                                                     GCCCGCGAAAAATTGACTGTCTACAGCTTCTGGGGGCCCACCATAAAC912                           AlaArgGluLysLeuThrValTyrSerPheTrpGlyProThrIleAsn                              290295300                                                                     CTGAGTGAACCCAACGCCTCCGAGGGGACTGCAGTGACTGTGACTTGC960                           LeuSerGluProAsnAlaSerGluGlyThrAlaValThrValThrCys                              305310315320                                                                  GCGGCCGGAGCCCGCGTCCAGGTCATGCTGGAGGGACTTCCGGCCGCG1008                          AlaAlaGlyAlaArgValGlnValMetLeuGluGlyLeuProAlaAla                              325330335                                                                     GCCCCTGGACAGCCTGCCCAGTTTCAGCTAAACGCCACCGAGATGGAC1056                          AlaProGlyGlnProAlaGlnPheGlnLeuAsnAlaThrGluMetAsp                              340345350                                                                     GACAGGCGCAGCTTCTTCTGCAATGCCACCCTCGAGGTGGATGGGGAG1104                          AspArgArgSerPhePheCysAsnAlaThrLeuGluValAspGlyGlu                              355360365                                                                     ACCTTACACAGGAACAGCAGCGTCCAGTTGCGTGTCCTGTACGGTCCC1152                          ThrLeuHisArgAsnSerSerValGlnLeuArgValLeuTyrGlyPro                              370375380                                                                     AAGATTGACCAAGCCAAATGTCCCCAGCGCTTGACGTGGAAAGAGAAA1200                          LysIleAspGlnAlaLysCysProGlnArgLeuThrTrpLysGluLys                              385390395400                                                                  ACTACCCATGTCCTGCAGTGCCAGGCTCGGGGCAACCCGGACCCCCAG1248                          ThrThrHisValLeuGlnCysGlnAlaArgGlyAsnProAspProGln                              405410415                                                                     ATGCACTGTTTTCACGAAGGCTCCCACGTCGAGCTGCCTATCGGGGTC1296                          MetHisCysPheHisGluGlySerHisValGluLeuProIleGlyVal                              420425430                                                                     CCATTCTTCGTCAGGTTAAACTATACTGGTACCTATGCCTGCAAGGCG1344                          ProPhePheValArgLeuAsnTyrThrGlyThrTyrAlaCysLysAla                              435440445                                                                     TCCAGCTCACGAGGCGTACACACTGTGACTGTGGTGATGAACGTTCAG1392                          SerSerSerArgGlyValHisThrValThrValValMetAsnValGln                              450455460                                                                     GATCGGAACCTCCGCGCTGTCAACATCGTCCTGGGGGTGTTAGCGATC1440                          AspArgAsnLeuArgAlaValAsnIleValLeuGlyValLeuAlaIle                              465470475480                                                                  TTGGGCGTGGTGACTACCGTCGCAGCCTTACTGCACGTCTTCGGGGTG1488                          LeuGlyValValThrThrValAlaAlaLeuLeuHisValPheGlyVal                              485490495                                                                     CAGAAGCGGAGTGACACCTACCGTGTGAACCAGGGGAGCACTTGGTTA1536                          GlnLysArgSerAspThrTyrArgValAsnGlnGlySerThrTrpLeu                              500505510                                                                     CCCCTGACGTCTAGGCAGCCCGAAGAGGCTGTGGGGGAGAATCCATCC1584                          ProLeuThrSerArgGlnProGluGluAlaValGlyGluAsnProSer                              515520525                                                                     TGAGCTTCCAGCTGGGATGATGGGGGTTTGGCTGTATCCCGGGGTTCCACACCAATAAAG1644              CCCTCAAACCCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA1693                         (2) INFORMATION FOR SEQ ID NO:119:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1404 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:119:                                     CAATTCGGAACGAGGTTCCCGGAGCGCGTGGAGCTGGCGCCCCTGCCCCCCTGGCAGCCC60                GTCGGGGAGGACCTCACCCTGYGYTGCCAGGTGGTGGGCGGGCAGGCCCGRGCCCGGCTC120               TCGGTGCTGCTGCTCCGCGGGGAGGAGGAGCTGAGCCGGCAGCCGGCGCTGGGGGAGCCC180               GTGGAGGTCACCGCCACGGTGCGGGCCGACAGAAGCGACCACGGCGCCAATTTCTCCTGC240               GTCACGGAGCTGGACCTGCGGCCCCTGGGCTTGGAGTTGTTCAAGAACGTCTCGGCCCCC300               AGGCAGCTGCAAACGTTCGTCCTGCCCCTGACTCCTCCGAGCCTCGCCGTCTTCCCGCTC360               TTGGAGGTGGGAACTTCGTGGCCGGTGGTCTGCCACCTGTCCGGCCTGTTCCCGGCCTCC420               GCGGCCCAGGTCCGGTTGGCGCTGGGAGACCAGATGCTGAATCCCCAAGTCACGAGGGAC480               GGGGACGCGCTCAACGCGACGGCCATAGTCACAGAGCGGTCAGGCCACGAGGGAGCGCGC540               GAGGTGGTCTGCAGCGTGACCCTGGCCGGCCGGAGTCGCGAGGCCCGGAAGAACGTGACC600               GTCTATAGCTTCCTAGGGCCATTTCTGAACCTGAGCGAGCCCATCGCCACCGAGGGGTCC660               AGCGTGACTGTGACTTGCACGGCCGGGGCCCGGGTCCAGCTCGTGCTGGACGGGGTTCGG720               GCCGCGGCTCCGGGGCAGCCCGTCCACTTGCAGCTAAATGCCACCGAGCGCGACGACGGG780               CGCAGCTTCTCGTGCGGCGCCACCCTCCAGGTGGACGGCCACTTTGTGCACCGCAACAGG840               AGCGCCCGCCTGCGTGTCCTGTACGGCCCCAGGATTGACCGGGCCACGTGTCCCCAGCAC900               GTNGCGTGGAGAGAAAGGACGACCCACGTCCTGCATTGCGAGGCTCGCGGCAACCCGGCC960               CCCCAGCTGAGGTGTTTGGAAGAGGGCTCCAGGCGCGAGGTGCCGGTCGGCGTCCCGTTC1020              CTCGTCCAGTTAAACTACAGTGGCACGTWTCGCTGCCAGGCGGCCAGCCCGCGGGGCACG1080              GACACCCTGGTCGTGGTGATGGACGTTCAAGGTATGAACTCCTCGACCGTCAGCATCGTC1140              CTAGGAGTGCTGGTCACCCTGGGCCTGGTGGCCGTTGCAGCGGCCTCCCTGTACATCTTC1200              GGGGTGCAGAAACGGAGCGGAAGCTACCCGGTGAAGCAGCCAGGCCCCTCGCTGCCCCTC1260              CAGTCCATGCAGCCCGAGGAAGGGGATGGCGGACAGCCGTCCTGAACCGCGGGCAGAAGT1320              CGGCGGGGCCTTCGGCAGTCTACCCCCGGGTTCCGTACCAATAAAGCTTCTAAACCCCCC1380              AAAAAAAAAAAAAAAAAAAAAAAA1404                                                  (2) INFORMATION FOR SEQ ID NO:120:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 434 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:120:                                     GlnPheGlyThrArgPheProGluArgValGluLeuAlaProLeuPro                              151015                                                                        ProTrpGlnProValGlyGluAspLeuThrLeuXaaCysGlnValVal                              202530                                                                        GlyGlyGlnAlaArgAlaArgLeuSerValLeuLeuLeuArgGlyGlu                              354045                                                                        GluGluLeuSerArgGlnProAlaLeuGlyGluProValGluValThr                              505560                                                                        AlaThrValArgAlaAspArgSerAspHisGlyAlaAsnPheSerCys                              65707580                                                                      ValThrGluLeuAspLeuArgProLeuGlyLeuGluLeuPheLysAsn                              859095                                                                        ValSerAlaProArgGlnLeuGlnThrPheValLeuProLeuThrPro                              100105110                                                                     ProSerLeuAlaValPheProLeuLeuGluValGlyThrSerTrpPro                              115120125                                                                     ValValCysHisLeuSerGlyLeuPheProAlaSerAlaAlaGlnVal                              130135140                                                                     ArgLeuAlaLeuGlyAspGlnMetLeuAsnProGlnValThrArgAsp                              145150155160                                                                  GlyAspAlaLeuAsnAlaThrAlaIleValThrGluArgSerGlyHis                              165170175                                                                     GluGlyAlaArgGluValValCysSerValThrLeuAlaGlyArgSer                              180185190                                                                     ArgGluAlaArgLysAsnValThrValTyrSerPheLeuGlyProPhe                              195200205                                                                     LeuAsnLeuSerGluProIleAlaThrGluGlySerSerValThrVal                              210215220                                                                     ThrCysThrAlaGlyAlaArgValGlnLeuValLeuAspGlyValArg                              225230235240                                                                  AlaAlaAlaProGlyGlnProValHisLeuGlnLeuAsnAlaThrGlu                              245250255                                                                     ArgAspAspGlyArgSerPheSerCysGlyAlaThrLeuGlnValAsp                              260265270                                                                     GlyHisPheValHisArgAsnArgSerAlaArgLeuArgValLeuTyr                              275280285                                                                     GlyProArgIleAspArgAlaThrCysProGlnHisValAlaTrpArg                              290295300                                                                     GluArgThrThrHisValLeuHisCysGluAlaArgGlyAsnProAla                              305310315320                                                                  ProGlnLeuArgCysLeuGluGluGlySerArgArgGluValProVal                              325330335                                                                     GlyValProPheLeuValGlnLeuAsnTyrSerGlyThrXaaArgCys                              340345350                                                                     GlnAlaAlaSerProArgGlyThrAspThrLeuValValValMetAsp                              355360365                                                                     ValGlnGlyMetAsnSerSerThrValSerIleValLeuGlyValLeu                              370375380                                                                     ValThrLeuGlyLeuValAlaValAlaAlaAlaSerLeuTyrIlePhe                              385390395400                                                                  GlyValGlnLysArgSerGlySerTyrProValLysGlnProGlyPro                              405410415                                                                     SerLeuProLeuGlnSerMetGlnProGluGluGlyAspGlyGlyGln                              420425430                                                                     ProSer                                                                        __________________________________________________________________________

We claim:
 1. A humanized ICR 1.1 antibody.
 2. A humanized ICR 8.1antibody.
 3. A humanized ICR-8.1 antibody comprising heavy chainvariable region miHuVHI and light chain variable region HuVK.